AU2004232964A1

AU2004232964A1 - Protected monomers

Info

Publication number: AU2004232964A1
Application number: AU2004232964A
Authority: AU
Inventors: Muthiah Manoharan
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2003-04-17
Filing date: 2004-04-16
Publication date: 2004-11-04
Anticipated expiration: 2024-04-16
Also published as: AU2004232964B2; JP2007525453A; WO2004094345A2; EP1625138A2; EP1625138A4; WO2004094345A8; JP4991288B2; CA2522349A1; WO2004094345A3

Description

WO 2004/094345 PCT/US2004/011822 Protected Monomers CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of United States Provisional Application No.: 60/463, 772, filed on April 17, 2003; United States Provisional Application No.: 60/465,802, filed on 5 April 25, 2003; United States Provisional Application No.: 60/493,986, filed on August 8, 2003; United States Provisional Application No.: 60/494,597, filed on August 11, 2003; United States Provisional Application No.: 60/506,341, filed on September 26, 2003; United States Provisional Application No.: 60/518,453, filed on November 7, 2003; United States Provisional Application No.: 60/469,612, filed on May 9, 2003; United States Provisional Application No.: 10 60/510,246, filed on October 9, 2003; United States Provisional Application No.: 60/510,318, filed on October 10, 2003; United States Provisional Application No.: 60/465,665, filed on April 25, 2003; International Application No.: [PCT/US04/07070], filed on March 8, 2004; International Application No.: [PCT/USO4/XXXXX], filed on April 5, 2004; International Application No.: [PCT/USO4/XXXXX], filed on April 9, 2004; and International Application 15 No.: [PCT/USO4/XXXXX], filed on even date herewith. The contents of all of these prior applications are hereby incorporated by reference in their entireties. TECHNICAL FIELD This invention relates to protected monomers for the synthesis of iRNA agents. BACKGROUND 20 Organic molecules containing two or more identical or similar functional groups (e.g., FGa, FGa', and FGa) are ubiquitous chemical species. Exposure of such a molecule to a particular reagent can be expected to produce products in which most or all of FGa, FGa

'

, and FGa" have reacted with the reagent, especially if the functional groups are located in similar steric or electronic environments on the molecule. When reaction at only one particular 25 functional group (e.g., FGa) is desired, it is necessary to block selectively the remaining functional groups (e.g., FG a' and FGa) with e.g., a "protecting group." A protecting group is a moiety that is temporarily attached to a potentially reactive site so as to prevent it from reacting. Once the reaction is complete at FGa, FGa' and FG'a" can be "deprotected," or restored to their original chemical form. 30 One example where the protecting group strategy has been utilized to provide functional group reaction selectivity is in the synthesis of oligoribonucleotides from individual 1 WO 2004/094345 PCT/US2004/011822 ribonucleotide monomer units. There are several chemically similar sites on the ribonucleoside monomers, e.g. the 2'-, 3'- and 5'- hydroxyl (OH) groups. However, the monomer subunits must be attached in a site-specific manner during RNA synthesis. Specifically, the 5'hydroxyl of one nucleoside or nucleotide chain is coupled to the 3' phosphate of a second nucleoside or 5 nucleotide chain. This requires functionalizing a site either on the growing chain or on the incoming base for attachment of the incoming monomer building block to the growing chain. To prevent the incoming monomer from attaching at the wrong site, the wrong sites must be blocked while the correct site is left open to react. This requires the use of the protecting group strategy. The protecting group must be stable during said reactions and yet must eventually be removed to 10 yield the original site. Additionally, the synthesis of oligonucleotides requires several sites to be protected and particular sites must be deprotected while others remain protected. These protecting groups, together as a set, are termed orthogonal protecting groups. These approaches have been widely used in the synthesis of DNA molecules. Phosphoramidite chemistry, so named for a functional group on the monomer building blocks, 15 has seen wide use in the synthesis of polynucleotides, see, e.g., U.S. 4,415,732. The phosphoramidite functional group allows for monomer-by-monomer synthesis in a relatively efficient manner. Synthesis is often performed on a solid phase, see, e.g., Caruthers et al. in U.S. 4,458,066. In this approach the growing DNA chain can be attached to an insoluble support via a linker, e.g., a long organic linker, which allows the growing DNA chain to be solubilized in the 20 solvent in which the support is placed. Phosphoramidite chemistry combined with solid phase synthesis has helped make directed synthesis of DNA widely available. Solid phase phosphoramidite oligonucleotide synthesis methods typically use a dimethoxytrityl protecting group for the 5' hydroxyl of nucleosides. The 3' hydroxyl position is protected with a phosphoramidite functionality. Synthesis generally proceeds from the 3' to the 25 5' of the ribose or deoxyribose sugar component of the phosphoramidite nucleoside. The 5' end of the growing chain is reacted with and coupled to the 3' phosphoramidite of the incoming base to form a phosphite triester intermediate. To insure that only one monomer is added in a round of synthesis the 5' hydroxyl of the newly added base is protected by a dimethoxytrityl group. Any unreacted 5' hydroxyls are "capped" off to stop the synthesis of this chain, which would be 30 one base short at the end of synthesis. Iodine oxidation is used after each coupling reaction to yield a more stable phosphotriester intermediate. Oxidation prevents the relatively unstable phosphite triester linkage from undergoing cleavage under the acidic conditions of subsequent synthesis steps. In order to add another monomer the 5' dimethoxytrityl protecting group of the newly added base must be removed or deprotected, e.g., by reaction with acidic solution to yield 2 WO 2004/094345 PCT/US2004/011822 a free 5' hydroxyl group which can be coupled to the next protected nucleoside phosphoramidite. This process is repeated until the desired sequence is synthesized. The use of the dimethoxytrityl group further prevents the use of other acid labile protecting groups. This is important for RNA synthesis because another hydroxyl group at the 2' 5 position must be protected. Thus, the synthesis of RNA presents additional problems, e.g., the need for a suitable 2' protecting group. Incorporation of a dimethoxytrityl protecting group strategy at the 5' position therefore prevents the successful use of acid labile groups for 2' protection during RNA synthesis. U.S. 5,889,136, has described protection strategies for use where the 2' position in ribonucleotides must be protected. 10 SUMMARY This invention relates to protected monomers for the synthesis of iRNA agents, methods of synthesis, and uses thereof. 3 WO 2004/094345 PCT/US2004/011822 In one aspect, this invention relates to protected monomers having a formula (I):

X

5 '

X

5 Si-O B x 5 Oy 1 0 X3 x 2 (I) 5 wherein, B is selected from the group consisting of: 4 WO 2004/094345 PCT/US2004/011822 R R 2

R

9 N

R

4

R

4' R Rio -N R 8 N R 1 1 N 12 N N R N N R, R 1 N 3 N R6 N X N N R16 R14 N N N N N N R 17 N N R13 Y 0 NH FR19 R20 21 R 24 N NIIR 25N X N 23 R 26 I R 22 5 WO 2004/094345 PCT/US2004/011822 anthracenyl, pyrenyl,

R

53 RR54 R46 R 51, R 54 Q'Q R52

R

55 ""N N R48 R R62 O R61 4NR" '" Q ivQ n Reo 9 R15R64 5 66 RS59 Rea8 Re R6 R 72 an iv Q R 6 R R70 R69 5 6 WO 2004/094345 PCT/US2004/011822

X

2 is an ortho ester protecting group, hydrogen, ethers, alkyl ethers, esters, halogens, protected amines, or protected hydroxyl moieties; 5 X3 is -O-P(OR 27

)N(R

28

)

2 or -O-L-R 29

X

5 ', x 5 ", X 5 ' include at least one alkoxy or siloxy substituent;

R

1 is hydrogen or Ci-C 4 alkyl;

R

2 is hydrogen, C 1

-C

4 alkyl, or C 2

-C

6 alkenyl optionally substituted with hydroxy, or C(O)NIHRa; 10 R 3 is hydrogen, halo, C 1

-C

4 alkyl, CI-C 4 thioalkoxy, NH 2 , NM b, or NRbR;

R

4 when taken together with R 4 ' forms oxo, or R 4 when taken together with R s forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

4' when taken together with R 4 forms oxo, or is 0-;

R

5 is hydrogen, C 1

-C

4 alkyl, or when taken together with R 4 forms a double bond 15 between the carbon and nitrogen atoms to which they are attached;

R

6 is hydrogen, halo, NH 2 , NFRb, or NRbRc;

R

7 is an unshared electron pair, or C 1

-C

4 alkyl;

R

8 when taken together with R 9 forms a double bond between the carbon and nitrogen atoms to which they are attached, or R when taken together with R 1 " forms a double bond 20 between the carbon and nitrogen atoms to which they are attached;

R

9 is hydrogen, C 1

-C

4 alkyl, or when taken together with R 8 forms a double bond between the carbon and nitrogen atoms to which they are attached; R1 0 is hydrogen or is absent; R" is hydrogen, C 1

-C

4 alkyl, or when taken together with R 8 forms a double bond 25 between the carbon and nitrogen atoms to which they are attached;

R

1 2 is hydrogen, formyl, or C 1

-C

4 alkyl optionally substituted with hydroxy or protected hydroxy;

R

13 and R 1 4 are each independently hydrogen or CI-C 4 alkyl;

R

15 is hydrogen, CT-C 4 alkyl, or (CH 2 )nCH(Rd)CH(NHRe)(COORg); 30 R is hydrogen or C 1

-C

4 alkyl;

R

17 is halo, NH 2 , NIR , or NRbR;

R

18 is cyano, C(=NH)NH 2 , or CH 2 NH(Rh);

R

19 is hydrogen, or C 1

-C

4 alkyl;

R

20 is: 7 WO 2004/094345 PCT/US2004/011822 (i) hydrogen; (ii) hydroxy or protected hydroxy; (iii) C 1

-C

4 alkoxy optionally substituted with COOR!; or (iv) C 1

-C

4 alkyl optionally substituted with hydroxy and/or COOR f , NH 2 , NHR m, or 5 CONH2;

R

21 is hydrogen, or when taken together with R 23 forms a double bond between the carbon atoms to which they are attached;

R

2 2 is hydrogen;

R

23 is hydrogen, or when taken together with R 21 forms a double bond between the 10 carbon atoms to which they are attached;

R

24 and R 25 are each, independently, hydrogen or C 1

-C

4 alkyl;

R

26 is (CH 2 )nCH(Rd)CH(NHIRe)(COOR);

R

27 is C 1

-C

6 alkyl optionally substituted with cyano, or C 2

-C

6 alkenyl;

R

28 is Cl-C 10 alkyl; 15 R 29 is a liquid or solid phase support reagent; Q is N or CR 44 ; Q' is N or CR 45 ; Q" is N or CR 47 ; Q"' is N or CR 49 ; 20 QiV is N or CR 50 ;

R

44 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NRbRc, C 1

-C

6 alkyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 forms -OCH 2 0-;

R

4 5 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb , or NRbRo, C 1

-C

6 25 alkyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3 -Cs heterocyclyl, a ligand, a tethered ligand, or when taken together with R 44 or R 46 forms -OCH20-;

R

46 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NRbRc, C 1

-C

6 alkyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3 -Cs heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 or R 47 forms -OCH 2 0-; 30 R 47 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NIIRb, or NRbR ,

CI-C

6 alkyl, C 6

-C

10 aryl, C 6

-C

10 heteroaryl, C 3 -Cs heterocyclyl, a ligand, a tethered ligand, or when taken together with R 46 or R 48 forms -OCH20-; 8 WO 2004/094345 PCT/US2004/011822

R

4 " is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbRc ,

C,-C

6 alkyl, C 6

-C

10 aryl, C 6

-C

10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 47 forms -OCH20-;

R

49

R

50 , R 51 , R 52 , R 5 3 , R 54 , R 57 s , R 5 , R 5 9 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , 5 R 70 , R 71 , and R 72 are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NIIRb , or NRbR ,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6

-C

10 aryl, C 6

-C

10 heteroaryl, C 3 Cs heterocyclyl, NC(0)R 7 , or NC(O)R;

R

55 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NRb, or NRbR, C 1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 0 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, NC(0)RI 7 , or NC(0)R, 10 or when taken together with R 56 forms a fused aromatic ring which may be optionally substituted;

R

56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NIRb, or NRbR ,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 0 aryl, C 6

-C

10 heteroaryl, C 3

-C

8 heterocyclyl, NC(O)R 7 , or NC(O)R, or when taken together with R 55 forms a fused aromatic ring which may be optionally 15 substituted; X is O, S, or Se; YisOorS; L is -C(O)(CH 2 )qC(O)-, or -C(O)(CH2)qS-; Provided that R', R 2 , and R cannot all be hydrogen; further provided that when R s is 20 hydrogen, R 6 cannot be NH 2 , NH(protecting group), or NH(iBu); further provided that when R 12 is hydrogen and R 8 and R" 1 together form a double bond between the carbon and nitrogen atoms to which they are attached, R 9 and R 1 0 cannot both be hydrogen; further provided that when X and Y are O, R 1 9 is hydrogen, and R 21 and R 23 together form a double bond between the carbon atoms to which they are attached, R 20 cannot be hydrogen or CH 3 ; 25 R Ris glycinyl, threonyl, or norvalyl, each of which may optionally be partially or fully protected;

R

b is C 1

-C

6 alkyl or a nitrogen protecting group; Rc is C 1

-C

6 alkyl; Rd is hydrogen, hydroxy, protected hydroxy, or OOH; 30 Re is hydrogen, a nitrogen protecting group, or COORg; R is hydrogen, or Ci-C 6 alkyl;

R

g is CI-Clo alkyl; 9 WO 2004/094345 PCT/US2004/011822 R" is hydrogen, or

R

RkO R, RO R and Rj when taken together forms a double bond between the carbon atoms to which they are attached, or R' and Rj when taken together form -0- between the carbon atoms to which 5 they are attached;

R

k and R' are each, independently, hydrogen, a hydroxyl protecting group,a sugar, or a fully or partially protected sugar;

R

m is CI-C 4 alkyl optionally substituted with COOH; Ro is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH 2 , 10 NHR b , or NRbR e,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6

-C

10 aryl, C 6

-C

10 heteroaryl, C 3

-C

8 heterocyclyl, NC(0)R" 7 , or NC(0)Ro; n is 1-4; and q is 0-4. 10 WO 2004/094345 PCT/US2004/011822 In another aspect, this invention relates to protected monomers having a formula (I): X5'

X

5 -Si - B xO X3

X

2 (I) wherein, 5 B is selected from the group consisting of: 11 WO 2004/094345 PCT/US2004/011822

R

1

R

2

R

9 N

R

4 R4' R R7 10 -N R 8 N N> N NR 1 N NN N 12 R3 R N N R 15 0 01 RIB 1

R

1 4 XN NN R N N4N NR7 N R13 1 y 0 NH R19 N R 2 0 21 R 2 4 N R25 S N " R23,and

R

26 HN N X N 0 2 22 5 X 2 is an ortho ester protecting group, hydrogen, ethers, alkyl ethers, esters, halogens, protected amines, or protected hydroxyl moieties;

X

3 is -O-P(OR 27

)N(R

28

)

2 or -O-L-R29

X

5 ', X s 5 ", X s' include at least one alkoxy or siloxy substituent;

R

1 is hydrogen or Cl-C 4 alkyl; 10 R 2 is hydrogen, C 1

-C

4 alkyl, or C 2

-C

6 alkenyl optionally substituted with hydroxy, or C(O)NZHRa;

R

3 is hydrogen, C 1

-C

4 alkyl, or C 1

-C

4 thioalkoxy, NIH 2 , NHR b, or NRbR;

R

4 when taken together with R 4 forms oxo, or R 4 when taken together with R s forms a double bond between the carbon and nitrogen atoms to which they are attached; 12 WO 2004/094345 PCT/US2004/011822

R

4 ' when taken together with R 4 forms oxo, or is O-;

R

5 is hydrogen, CI-C 4 alkyl, or when taken together with R 4 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

6 is hydrogen, NH 2 , NHR b , or NR bR; 5 R 7 is an unshared electron pair, or CI-C 4 alkyl; R when taken together with R 9 forms a double bond between the carbon and nitrogen atoms to which they are attached, or R 8 when taken together with R 1 1 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

9 is hydrogen, C 1

-C

4 alkyl, or when taken together with R 8 forms a double bond 10 between the carbon and nitrogen atoms to which they are attached;

R

I0 is hydrogen or is absent;

R"

1 is hydrogen, C 1

-C

4 alkyl, or when taken together with R 8 forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

1 2 is hydrogen, formyl, or Ci-C 4 alkyl optionally substituted with hydroxy or protected 15 hydroxy;

R

13 and R 14 are each independently hydrogen or C 1

-C

4 alkyl;

R

1 5 is hydrogen, CI-C 4 alkyl, or (CH 2 )nCH(Rd)CH(NHRe)(COOR);

R

1 6 is hydrogen or C 1

-C

4 alkyl;

R

17 is halo, NH 2 , NHIRb, or NRbR; 20 R 18 is cyano, C(=NH)NH 2 , or CH 2 NH(Rh);

R

1 9 is hydrogen, or C 1

-C

4 alkyl;

R

2 0 is: (i) hydrogen; (ii) hydroxy or protected hydroxy; 25 (iii) CI-C 4 alkoxy optionally substituted with COORf; or (iv) C 1

-C

4 alkyl optionally substituted with hydroxy and/or COOR', NH 2 , NHR m , or

CONHI

2 ;

R

2 1 is hydrogen, or when taken together with R 23 forms a double bond between the carbon atoms to which they are attached; 30 R 22 is hydrogen; R23 is hydrogen, or when taken together with R 21 forms a double bond between the carbon atoms to which they are attached;

R

24 and R 2 5 are each, independently, hydrogen or C 1

-C

4 alkyl;

R

26 is (CH2)nCH(Rd)CH(NHR)(COOR"); 13 WO 2004/094345 PCT/US2004/011822 R27 is C 1

-C

6 alkyl optionally substituted with cyano, or C 2

-C

6 alkenyl;

R

28 is C 1

-C

1 0 alkyl;

R

29 is a liquid or solid support reagent; X is O, S, or Se; 5 YisOorS; L is -C(0)(CH 2 )qC(O)-, or -C(O)(CH 2 )qS-; Provided that R 1 , R 2 , and R 3 cannot all be hydrogen; further provided that when R 5 is hydrogen, R 6 cannot be NH2 or NH(iBu); further provided that when R 12 is hydrogen and R 8 and R" together form a double bond between the carbon and nitrogen atoms to which they are 10 attached, R 9 and R1 0 cannot both be hydrogen; further provided that when X and Y are O, R 19 is hydrogen, and R 2 ' and R 2 3 together form a double bond between the carbon atoms to which they are attached, R 20 cannot be hydrogen or CH 3 ; Ra is glycyl, threonyl, or norvalyl, each of which is optionally partially or fully protected;

R

b is C 1

-C

6 alkyl or a nitrogen protecting group; 15 RC is CI-C 6 alkyl; Rd is hydrogen, hydroxy, protected hydroxy, or OOH; Re is hydrogen, a nitrogen protecting group, or COOR; R is hydrogen, or CI-C 6 alkyl; R is C 1 -Clo alkyl; 20 R h is hydrogen, or

R

RkO RO R and Rj when taken together forms a double bond between the carbon atoms to which they are attached, or R i and Rj when taken together form -0- between the carbon atoms to which they are attached; 25 R k and R 1 are each, independently, hydrogen, a hydroxyl protecting group, a sugar, or a;

R

m is CI-C 4 alkyl optionally substituted with COOH; n is 1-4; and 14 WO 2004/094345 PCT/US2004/011822 q is 0-4. In a further aspect, this invention relates to protected monomers having a formula (I):

X

5

,

'

X

"

-Si-O B 15~ x X3 X 2 (I) 5 wherein, B is selected from the group consisting of: anthracenyl, pyrenyl, R53 R54 R46 R51 R 'Q "'Q Q . Q IQ i v N/R 5 2 Q g i v / R 5 5

R

4 8 5

R

5 6 I15 15 WO 2004/094345 PCT/US2004/011822

R

6 2 R 61 R 63 nI IVQ N, and R60 R57 R64

R

6 7 it5815, 6 5

R

66 R59 R58 R6 R6 R72 uQ iv Q R 68 R R1 70 R69 16 WO 2004/094345 PCT/US2004/011822

X

2 is an ortho ester protecting group, hydrogen, ethers, alkyl ethers, esters, halogens, protected amrnines, or protected hydroxyl moieties;

X

3 is -O-P(OR 27

)N(R

28

)

2 or -O-L-R 29 ; 5 X 5 ', X 5 ", X 5 "' include at least one alkoxy or siloxy substituent;

R

17 is halo, NH 2 , NHR b, or NRbR;

R

27 is C 1

-C

6 alkyl optionally substituted with cyano, or C 2

-C

6 alkenyl;

R

28 is C 1

-C

10 alkyl;

R

29 is a liquid or solid phase support reagent; 10o Q is N or CR 44 ; Q' is N or CR 45 ; Q" is N or CR 4 7 ; Q"' is N or CR 4 9 ; QiV is N or CRs°; 15 R is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbRc, C 1 -C6 alkyl, C 6 -Co10 aryl, C 6 -Clo heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 4 5 forms -OCH 2 0-;

R

45 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR ,

C

1

-C

6 alkyl, C 6 -Clo aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when 20 taken together with R 44 or R 46 forms -OCH 2 0-;

R

46 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR ,

C

1

-C

6 alkyl, C 6

-C

1 0 aryl, C 6

-C

10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 or R 47 forms -OCH 2 0-;

R

47 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b, or NRbRC ,

CI-C

6 25 alkyl, C 6

-C

10 aryl, C 6

-C

1 0 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 46 or R 48 forms -OCH20-;

R

4 8 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR ,

CI-C

6 alkyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 47 forms -OCH20-; 30 R 4 9

R

50 , Rs 1 , R 52 , R 5 3 , R 5 4 , R 5 7 , R 5 8 , R 5 9 , R 6 0 , R 6 1 , R 6 2 , R 6 3 , R 64 , R 6 5 , R 6 6 , R , R 6 9 ,

R

7 0 , R 71 , and R 72 are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NHI 2 , NHRb, or NRbRc ,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6 -Cl 0 aryl, C 6 -Co 10 heteroaryl, C 3 C 8 heterocyclyl, NC(O)R 7 , or NC(O)R; 17 WO 2004/094345 PCT/US2004/011822

R

55 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR c, CI -C 6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 0 aryl, C 6

-C

10 heteroaryl, C 3 -C8 heterocyclyl, NC(O)R 7 , or NC(O)Ro, or when taken together with R 56 forms a fused aromatic ring which may be optionally substituted; 5 R 56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NRbR, C 1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, NC(O)R" 7 , or NC(O)Ro, or when taken together with R 55 forms a fused aromatic ring which may be optionally substituted; L is -C(O)(CH 2 )qC(O)-, or -C(O)(CH 2 )qS-; 10 R b is C 1

-C

6 alkyl or a nitrogen protecting group; RC is C 1

-C

6 alkyl; Ro is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH 2 ,

NR

b , or NRbR e ,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 0 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl,

NC(O)R

7 , or NC(O)Ro; 15 n is 1-4; and q is 0-4. 18 WO 2004/094345 PCT/US2004/011822 Embodiments can include one or more of the following features. B can be: R1

R

2 N N R3 N 5 B can be:

R

4

R

4'

R

7 R" N R6 B can be:

R

9 R o- N R' R 1 N R12 X N I 10 19 WO 2004/094345 PCT/US2004/011822 B can be:

R

15 RR14 N N "N B can be: 5 0 R18 OR R16 R17N R NN B can be: Y 20 19 R R N (R 21

R

23 X N x~ RN22 10 20 WO 2004/094345 PCT/US2004/011822 B can be: 0

R

2 4 R 2 5 ",N NR 0 5 B can be: NH NI R26HN N B can be:

R

4 6 QQ R48 I 10 21 WO 2004/094345 PCT/US2004/011822 B can be:

R

51 Rs Q Qv R52 N B can be:

R

53 R54 fit Q iv R// 55 N

+R

56 5 B can be: R62 O R61 R60 Re " R59 R58 10 22 WO 2004/094345 PCT/US2004/011822 B can be: R63 ,,, Q ivQ R64 R67 R65 R 5 B can be: R 72 fit Q IV Q R 6 R 7

R

7 0

R

6 9 B can be anthracenyl. B can be pyrenyl. 10 R 28 can be isopropyl.

X

5' , X 5' , and X 5 "'" can be any combination of the following formula: 23 WO 2004/094345 PCT/US2004/011822 PAP

CH

3 ,- 7

H

3 C-Si Si

CH

3 3 3 and O 0 ( 3 3

X

5 'and X 5 ' " can be siloxy and X s 5 ' can be cycloalkoxy. The orthoester group can have formula (III):

O-

32 R320

OR

3 1 5 (III) 24 WO 2004/094345 PCT/US2004/011822

R

3 and R 32 can be the same or different and can be any combination of the following formulae: CI C1 OCH3 CN 0 Cl CI O Cl C R33 R34 34 R 33 3 5 R 33 R 3

R

33 0R 3 3 or 0O R38 5 wherein R 33 , R 34 , R 35 , R 3 6 , and R 3 7 is a compatible ligand, or hydrogen, or halogen, alkyl, or cyano substituent, and R 38 is compatible ligand. 25 WO 2004/094345 PCT/US2004/011822 The orthoester can be: O

H

3 C O 0 O

CH

3 0 0 O O

R

29 can be a fluoride-stable polystyrene based solid support or PEG. 5 X 2 can be -OC[OCH 2

CH

2 OC(0)CH 3

]

2 ; R 27 can be CH 3 ; R 2 8 can be (CH 3 )2CH-; X5' and X5" are trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be:

R

I N R 2 N N R3

X

2 can be -OC[OCH 2

CH

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 2 8 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be: 10

R

4

R

4' R

R

5 N N N) 6 6 R NN 26 WO 2004/094345 PCT/US2004/011822

X

2 can be -OC[OCH 2

CH

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be:

R

9 10 1 Ro N R R 1 N R 12 X N 5 X 2 can be -OC[OCH 2

CH

2 OC(O)CH3] 2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: R O R 15 0 R 1 3 N 1 13

X

2 can be -OC[OCH 2 CH20C(O)CH 3

]

2 ; R 2 7 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and 10 X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: O R18

R'

7 R 7 N N 27 WO 2004/094345 PCT/US2004/011822

X

2 can be -OC[OCH 2

CH

2 OC(O)CH3] 2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: Y R 20 R1 , R 21 N R R 2 3 X N

X

2 can be -OC[OCH 2

CHI

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and 5 X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: O

R

2 4 "

R

2 5

X

2 can be -OC[OCH 2

CH

2 OC(0)CH 3

]

2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: 10 NH N R26HN N I28 28 WO 2004/094345 PCT/US2004/011822

X

2 can be -OC[OCH2 20C()CH32; 7 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be: R46 Q R48 I

X

2 can be -OC[OCH 2

CH

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R can be (CH 3

)

2 CH-; X5' and 5 X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be: X2 can be -OC[OCH2CH20C(O)CH3]2; R 2 7 can be CH3; R 8 can be (CH3)2CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be:

R

5 3 Q QIV R 5 4 Qiv// R 5 'N R56 10 29 WO 2004/094345 PCT/US2004/011822

X

2 can be -OC[OCH 2 CH20C(O)CH 3

]

2 ; R 2 7 can be CH 3 ; R 2 8 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be:

R

62 O R N R 60 R 57 R59 R 5 5X 2 can be -OC[OCH 2

CH

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be: R 63 ip iv R64,1 . R67 R4 R Re65 R66

X

2 can be -OC[OCH 2

CH

2

OC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 8 can be (CH 3

)

2 CH-; X5' and 10 X5" can be trimethylsiloxy; X5"' can be cyclododecyloxy; and B can be:

R

7 2 f" Qo iv Q R 68 R71 R7O R 69 30 WO 2004/094345 PCT/US2004/011822

X

2 can be -OC[OCH 2 CH20C(O)CH 3

]

2 ; R 27 can be CH 3 ; R 8 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be anthracenyl.

X

2 can be -OC[OCH 2

CHOC(O)CH

3

]

2 ; R 27 can be CH 3 ; R 2 8 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be pyrenyl. 5 B can be an unusual or universal base that can be selected from: 2-aminoadeninyl, 2 methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl, N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl, N6-(cis-hydroxyisopentenyl)adeninyl, 2-methylthio-N6 (cis-hydroxyisopentenyl) adeninyl, N6-glycinylcarbamoyladeninyl, N6 threonylcarbamoyladeninyl, 2-methylthio-N6-threonyl carbamoyladeninyl, N6-methyl-N6 10 threonylcarbamoyladeninyl, N6-hydroxynorvalycarbamoyladeninyl, 2-methylthio-N6 hydroxynorvalyl carbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methylcytosinyl, 5 methylcytosinyl, 2-thiocytosinyl, 5-formylcytosinyl, N4-methylcytosinyl, 5 hydroxymethylcytosinyl, 1-methylguaninyl, N2-methylguaninyl, 7-methylguaninyl, N2,N2 dimethylguaninyl, 15 NH COOH N

H

2 N N N H 31 WO 2004/094345 PCT/US2004/011822

NHCOOCH

3 NHCOOCH 3 NHCOOCH 3

H

3 COOC

H

3 COOC OH

H

3 COOC OOH 0 0 0 /N NNf' N N

H

3 C N H 3

H

3 N'N N NN N-N N I I- CH 3 CH 3 CH 3

NH

2 HOOC OH O O 0 H3 NNNN

NH

3 HCH3 CH 3 o H HO HN N HCN N HO HO O NN NH NH HN HN

H

2 N N H 2 N N HO HO beta-galactosylO o beta-mannosyl O o HN NH HoNH2 0NH 0N NH 2 HNH HN N

H

2 N N N H 2 N H 2 N "N N 5 32 WO 2004/094345 PCT/US2004/011822 N2,7-dimethylguaninyl, N2,N2,7-trimethylguaninyl, 1-methylguaninyl, 7-cyano-7 deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl, dihydrouracilyl, 5 methyluracilyl, 1-methylpseudouracilyl, 2-thiouracilyl, 4-thiouracilyl, 5-methyl-2-thiouracilyl, 3 (3-amino-3-carboxypropyl)uracilyl, 5-hydroxyuracilyl, 5-methoxyuracilyl, uracilyl 5-oxyacetic 5 acid, uracilyl 5-oxyacetic acid methyl ester, 5-(carboxyhydroxymethyl)uracilyl, 5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonylmethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl, 5-aminomethyl-2-thiouracilyl, 5-methylaminomethyluracilyl, 5-methylaminomethyl-2 thiouracilyl, 5-methylaminomethyl-2-selenouracilyl, 5-carbamoylmethyluracilyl, 10o 5-carboxymethylaminomethyluracilyl, 5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl, 1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl, 5 carboxymethyluracilyl, 5-methyldihydrouracilyl, 3-methylpseudouracilyl, F H 3

NH

2

NH

2 0 2 N

H

3 C N N N N F &N ,,y CH O H O O OCH 3 O N0 NN H NN- N N ', BuHN N N- 02 H H N 15 33 WO 2004/094345 PCT/US2004/011822 O

CH

3

H

3 C CH3 N N N

CCH

3 , and CN

CH

3 X can be -OC[OCH 2 CH20C(O)CH 3

]

2 ; R 27 can be CH 3 ; R 28 can be (CH 3

)

2 CH-; X5' and X5" can be trimethylsiloxy; X5'" can be cyclododecyloxy; and B can be selected from any of 5 the unusual or universal bases described above.

X

2 can be fluoro. B can be: F

CH

3 F B can be substituted or unsubstituted (e.g., having one or more fluoro groups) aryl 10 attached to a tethered or untethered ligand. 34 WO 2004/094345 PCT/US2004/011822 In one aspect, this invention relates to this invention relates to a protected monomer having a formula (II): X5,' X' -Si- OB G 5 wherein B is selected from the group selected from: 35 WO 2004/094345 PCT/US2004/011822

R

1

R

2 R 9 N

R

4

R

4 ' R RioN R N N N N R 12 N N

R

3

NR

6 N N R X N R15 O O R18 N R

R

14 R N N R1311 Y ONNH N N R 17 N N 13 R o NH R19 R 24 2 N ",,,i R 21

N

/

N

/

R

2 3 26 0N RHN N X N 22 S- R2 2 36 WO 2004/094345 PCT/US2004/011822 anthracenyl, pyrenyl, R53

R

54 R46 R51 '"Q Q Q' Q Qiv 5iv R 55 °' R5 R48 R I

R

6 2 O 61 R63 ,,, Q iv, N and RR6

R

6 0 R 57 R64 R67 R59 ea 65 R66 R72 , Q v Q

R

6 8 R R 70 R 69 5 37 WO 2004/094345 PCT/US2004/011822

X

2 is an ortho ester protecting group, hydrogen, ethers, alkyl ethers, esters, halogens, protected amines, or protected hydroxyl moieties;

X

3 is -O-P(OR 27

)N(R

28

)

2 or-O-L-R29

X

5' , X 5 ", X 5 "' include at least one alkoxy or siloxy substituent; 5 G is NR 3 0 , S, or CW 2 ;

R

1 is hydrogen or C 1

-C

4 alkyl;

R

2 is hydrogen, C 1

-C

4 alkyl, or C 2

-C

6 alkenyl optionally substituted with hydroxy, or C(O)NHRa;

R

3 is hydrogen, halo, CI-C 4 alkyl, C 1

-C

4 thioalkoxy, NHI 2 , NHRb, or NRbRc; 10 R 4 when taken together with R 4' forms oxo, or R 4 when taken together with R s forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

4' when taken together with R 4 forms oxo, or is 0;

R

s is hydrogen, C 1

-C

4 alkyl, or when taken together with R 4 forms a double bond between the carbon and nitrogen atoms to which they are attached; 15 R is hydrogen, halo, NHi 2 , NHR b, or NRbRe;

R

7 is an unshared electron pair, or C 1

-C

4 alkyl; R8 when taken together with R 9 forms a double bond between the carbon and nitrogen atoms to which they are attached, or R 8 when taken together with R 11 forms a double bond between the carbon and nitrogen atoms to which they are attached; 20 R 9 is hydrogen, C 1

-C

4 alkyl, or when taken together with Rg forms a double bond between the carbon and nitrogen atoms to which they are attached;

R

1 0 is hydrogen or is absent;

R"

1 is hydrogen, C 1

-C

4 alkyl, or when taken together with R 8 forms a double bond between the carbon and nitrogen atoms to which they are attached; 25 R 12 is hydrogen, formyl, or C 1

-C

4 alkyl optionally substituted with hydroxy or protected hydroxy;

R

13 and R 14 are each independently hydrogen or C 1

-C

4 alkyl;

R

15 is hydrogen, CI-C 4 alkyl, or (CH 2 )nCH(Rd)CH(NHRe)(COORg);

R

16 is hydrogen or C 1

-C

4 alkyl; 30 R 17 is halo, NH 2 , NHR b, or NRbR;

R

18 is cyano, C(=NH)NH 2 , or CH 2 Ni(Rh);

R

19 is hydrogen, or CI-C 4 alkyl;

R

2 is: (i) hydrogen; 38 WO 2004/094345 PCT/US2004/011822 (ii) hydroxy or protected hydroxy; (iii) C 1

-C

4 alkoxy optionally substituted with COOR!; or (iv) C 1

-C

4 alkyl optionally substituted with hydroxy and/or COOR', NH 2 , NR m, or CONH2; 5 R 2 1 is hydrogen, or when taken together with R 23 forms a double bond between the carbon atoms to which they are attached;

R

2 2 is hydrogen;

R

23 is hydrogen, or when taken together with R 21 forms a double bond between the carbon atoms to which they are attached; 10 R 2 4 and R 25 are each, independently, hydrogen or C 1

-C

4 alkyl;

R

26 is (CH 2 )nCH(Rd)CH(NHRe)(COORg);

R

27 is C 1

-C

6 alkyl optionally substituted with cyano, or C 2

-C

6 alkenyl;

R

28 is C 1 -Clo 0 alkyl;

R

29 is a liquid or solid phase support reagent; 15 Ro 30 is CI-C 2 0 alkyl, C 2

-C

20 alkenyl, C 2

-C

2 0 alkynyl; C 3

-C

8 cycloalkyl; C 6

-C

1 2 aryl; 5-10 membered heteroaryl; C 7

-C

1 4 aralkyl; -C(O)-(CH 2 )s-C(O)-(ligand); -C(O)-(CH2)s-C(0)O-(ligand); -C(O)-O-(ligand); -C(O)-(CH2)s-N -; -C(O)-(CH2)s-NH-C(O)-(ligand); -C(O)-(CH2),-(ligand); -C(O)-NH-(ligand); -C(O)-(ligand); -(CH2)s-C(O)-(ligand); -(CH2)s-C(0)O-(ligand); -(CH2),-(ligand); 20 -(CH 2 ),-NH-; or -(CH 2 ),-NH-C(O)-(ligand); Q is N or CR 44 ; Q' is N or CR 45 ; Q" is N or CR 47 ; Q'" is N or CR 4 9 ; 25 QiV is N or CR 50 ;

R

44 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbRc, C 1

-C

6 alkyl, C 6

-C

10 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 .forms -OCH 2 0-;

R

45 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb , or NbR, CI-C 6 30 alkyl, C 6

-C

10 aryl, C 6

-C

1 0 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 44 or R 46 forms -OCH 2 0-; R4 6 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NRbR, C 1

-C

6 alkyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3

-C

8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 or R 47 forms -OCH 2 0-; 39 WO 2004/094345 PCT/US2004/011822

R

47 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NHI 2 , NHRb, or NRbRc, Ci-C 6 alkyl, C 6 -Co 10 aryl, C 6

-C

1 0 heteroaryl, C 3

-C

R

48 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b, or NRbRc, C1-C 6 5 alkyl, C 6

-C

1 0 aryl, C 6

-C

10 heteroaryl, C 3

-C

R

49

RO

50 , R 51 , R 52 , R 53 , R 54 , R 7 , R 58 , R 5 9 , R 60 , R 61 , R2 63 , R 64 , R 6 5,

R

66 , R 7 , R 68 , R 9 ,

R

7 0 , R 71 , and R 72 are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, N- 2 , NE b , or NRbR , CI-C 6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 o aryl, C 6

-C

10 heteroaryl, C 3 10 Cs heterocyclyl, NC(O)R 17 , or NC(O)R';

R

5 5 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR, C 1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6 -C1O aryl, C 6 -Clo heteroaryl, C 3

-C

8 heterocyclyl, NC(O)R 7 , or NC(O)Ro, or when taken together with R s 6 forms a fused aromatic ring which may be optionally substituted; 15 R 56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbRc, C,-C 6 alkyl, C 2

-C

6 alkynyl, C 6

-C

1 o aryl, C 6

-C

10 heteroaryl, C 3

-C

8 heterocyclyl, NC(O)R 7 , or NC(O)RO, or when taken together with R 55 forms a fused aromatic ring which may be optionally substituted; X is O, S, or Se; 20 Y is O or S; L is -C(O)(CH 2 )qC(O)-, or -C(O)(CH 2 )qS-;

R

a is glycinyl, threonyl, or norvalyl, each of which may optionally be partially or fully protected; Rb is C 1

-C

6 alkyl or a nitrogen protecting group; 25 RC is CI-C 6 alkyl; Rd is hydrogen, hydroxy, protected hydroxy, or OOH; Re is hydrogen, a nitrogen protecting group, or COOR; R is hydrogen, or C 1

-C

6 alkyl; R9 is C-Co alkyl; 30 R h is hydrogen, or 40 WO 2004/094345 PCT/US2004/011822 R. RkO R'O R' and Rj when taken together forms a double bond between the carbon atoms to which they are attached, or R and Rj when taken together form -0- between the carbon atoms to which they are attached; 5 R k and R' are each, independently, hydrogen, a hydroxyl protecting group, a sugar, or a fully or partially protected sugar;

R

m is C 1

-C

4 alkyl optionally substituted with COOH; Ro is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR ,

C

1

-C

6 alkyl, C 2

-C

6 alkynyl, C 6 -Co 10 aryl, C 6

-C

1 0 heteroaryl, C 3

-C

8 heterocyclyl, 10 NC(0)R 7 , or NC(0)Ro; n is 1-4; q is 0-4; s is 0-20. Embodiments can include one or more of the features described above and can further 15 include protected monomers in which R', R 2 , and R 3 cannot all be hydrogen; further provided that when R 5 is hydrogen, R 6 cannot be NH 2 NH(protecting group), or NH(iBu); further provided that when R 1 2 is hydrogen and R 8 and R" 11 together form a double bond between the carbon and nitrogen atoms to which they are attached, R 9 and R 1 0 cannot both be hydrogen; further provided that when X and Y are O, R1 9 is hydrogen, and R 21 and R 23 together form a double bond between 20 the carbon atoms to which they are attached, R 2 0 cannot be hydrogen or CH 3 ; and/or W can be fluoro. 41 WO 2004/094345 PCT/US2004/011822 In another aspect, this invention relates to a protected monomer having a first functionalized hydroxyl group, a second functionalized hydroxyl group, and a ligand, in which the first functionalized hydroxyl group, the second functionalized hydroxyl group, and the ligand 5 are linked to a carrier. The first functionalized hydroxyl group can have the formula: X5' 1 °-1 Xs"-0i-O Xr Preferred X5', X5", and X5'" include siloxy and alkoxy or cycloalkoxy. The second functionalized hydroxyl group can have the formula: Ok O I or

(R

2 8 )2N - OR27 L 10

R

28 is C1-C 1 0 alkyl, e.g., isopropyl; R 27 is CI-C 6 alkyl optionally substituted with cyano, or C2-C6 alkenyl, e.g., methyl, allyl and 2-cyanoethyl. L is a linker and * is a liquid or solid support reagent. The ligand can be a targeting group (e.g., a lipid, steroid, vitamin, carbohydrate, 15 polyamine, amino acid, peptide, peptide mimetic or cleaving molecule) or the ligand may be a diagnostic group (e.g., biotin, a fluorophore, an antibody or an antigen). The ligand can have a formula (G)C(=H)NIR n , in which G is -0-, -NH-, or -CH 2 -; H is O or NH; and R n is H, C 1

-C

6 alkyl, C 6 -C10 aryl, or C 5 -Co 10 heteroaryl. The ligand may also be linked to the carrier through a tether. The tether can be -C(O)-(CH 2 )s-C(O)-(ligand); 20 -C(0)-(CH2)s-C(0)O-(ligand); -C(O)-O-(ligand); -C(O)-(CH2)s-NT-; -C(0)-(CH2)s-NH-C(0)-(ligand); -C(0)-(CH2)s-(ligand); -C(O)-NH-(ligand); -C(O)-(ligand); -(CH2)s-C(0)-(ligand); -(CH2)s-C(0)O-(ligand); -(CH2)s-(ligand);

-(CH

2 )s-NH-; or -(CH 2 )s-NH-C(O)-(ligand). The carrier can be a a cyclic moiety and may also contain one or more heteroatoms (e.g., 25 nitogen, oxygen, or sulfur). In some embodiments, the ligand can be attached to the nitrogen 42 WO 2004/094345 PCT/US2004/011822 atom of the cyclic moiety. Preferably, the monomer contains only two functionalized hydroxyl groups. In some embodiments, the cyclic moiety can be: N

(CH

2 ) N R40 R41 5 in which

R

39 and R 4 0 are each independently hydrogen or when taken together form oxo;

R

41 is hydrogen or -C(R 42

)(R

43

)-(CH

2 ) u- ;

R

4 W and R 43 are each independently hydrogen or when taken together form oxo; 10 u is 1 or 2; the wavy line represents a point of attachment for a ligand or a tethered ligand; and the dotted lines represent points of attachment for the first and second functionalized hydroxyl groups. 43 WO 2004/094345 PCT/US2004/011822 In some embodiments, the cyclic moiety can be: ------ u(H 2 C) N * S in which 5 u is 1 or 2; the wavy line represents a point of attachment for a ligand or a tethered ligand; and the dotted lines represent points of attachment for the first functionalized hydroxyl group, the second functionalized hydroxyl group, and an unfunctionalized hydroxyl group, a protected hydroxyl group, or hydrogen. In a further aspect, this invention relates to iRNA agents, which incorporate one or more 10 of the monomers described herein. The invention also relates to methods of using the iRNA agents. In one aspect, this invention relates to a method of synthesizing a polymer, the method includes: providing a 5' protected first monomer, providing a second monomer, deprotecting the 5' moiety of the first monomer, arid reacting the 3' moiety of the second monomer with the 15 deprotected 5' monomer, thereby synthesing a polymer, provided that one of the monomers is a monomer as described herein. In preferred embodiments, the monomer as described herein is provided as the 3' terminal residue of the polymer or the 5' terminal residue of the polymer. In another aspect, this invention relates to a di-, tri, or polymeric molecule which comprises a 5' silyl protecting group described herein and a subunit comprising at least one of 20 the monomers described herein. In a further aspect, this invention relates to a method of making an iRNA agent, the method includes providing a first sequence and a second sequence which can form a duplex, which includes at least one monomer added by a method described herein. In some embodiments, the first and second sequences are between 15 and 30 nucleotides in length. 25 In one aspect, this invention relates to a method of modulating expression of a target gene, the method includes providing an iRNA agent comprising a monomer which includes a monomer described herein or which was incorporated by a method described herein. In some embodiments, the iRNA agent can be administered to a subject. 44 WO 2004/094345 PCT/US2004/011822 In another aspect, this invention relates to a pharmaceutical composition comprising an iRNA agent which includes a monomer described herein or made by a method described herein. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the 5 invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS FIG. 1 is a reaction scheme showing how the protected monomers can be incorporated into the terminal and internal positions of a growing chain of monomers. FIG. 2 is a list of substituents that may be present on silicon in OFG 1 . 10 FIG. 3 is a list of substituents that may be present on the C2'-orthoester group. FIG. 4 is a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide. FIG. 5 is a list of representative RRMS carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based RRMSs; panel 15 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 is succinate or phosphoramidate and R2 is H or a conjugate ligand. FIG. 6 is a structural representation of base pairing in psuedocomplementary siRNA 2 . FIG. 7 is a schematic representation of dual targeting siRNAs designed to target the HCV genome. 20 FIG. 8 is a schematic representation of psuedocomplementary, bifunctional siRNAs designed to target the HCV genome. DETAILED DESCRIPTION PROTECTED MONOMERS 25 Definitions As used herein, the term "halo" or "halogen" refers to any radical of fluorine, chlorine, bromine or iodine. The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1~C 12 alkyl indicates 30 that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl 45 WO 2004/094345 PCT/US2004/011822 moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms "arylalkyl" or "aralkyl" refer to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. 5 Examples of"arylalkyl" or "aralkyl" include benzyl, 9-fluorenyl, benzhydryl, and trityl groups. The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more double bonds. The sp 2 carbon may optionally be the point of attachment of the alkenyl group to another moiety. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl 10 groups. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. The sp 3 carbon may optionally be the point of attachment of the alkynyl group to another moiety. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The terms "alkylamino" and "dialkylamino" refer to -NH(alkyl) and -NH(alkyl) 2 15 radicals respectively. The term "aralkylamino" refers to a -N-NH(aralkyl) radical. The term "alkoxy" refers to an -O-alkyl radical, and the terms "cycloalkoxy" and "aralkoxy" refer to an O-cycloalkyl and O-aralkyl radicals respectively. The term "siloxy" refers to a R 3 SiO- radical. The term "mercapto" refers to an SH radical. The term "thioalkoxy" refers to an -S-alkyl radical. The term "alkylene" refers to a divalent alkyl (i.e., -R-), e.g., -CH2-, -CH 2

CH

2 -, and 20 CH 2

CH

2

CH

2 -. The term "alkylenedioxo" refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene. The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl. 25 The term "cycloalkyl" as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom can be substituted. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and 30 norbornyl. The term "heterocyclyl" refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if 46 WO 2004/094345 PCT/US2004/011822 monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, 5 piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl. The term "cycloalkenyl" as employed herein includes partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons, wherein any ring atom can be substituted. The cycloalkenyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon 10 carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of cycloalkenyl moieties include, but are not limited to cyclohexenyl, cyclohexadienyl, or norbornenyl. The term "heterocycloalkenyl" refers to a partially saturated, nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if 15 tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heterocycloalkenyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenyl include but are not limited to 20 tetrahydropyridyl and dihydropyran. The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or 25 tricyclic, respectively), wherein any ring atom can be substituted. The heteroaryl groups herein described may also contain fused rings that share a common carbon-carbon bond. The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur. The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, 30 heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents. The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, 47 WO 2004/094345 PCT/US2004/011822 halo, hydroxy, cyano, nitro, amino, SO3H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)nalkyl (where n is 0-2), S(O), aryl (where n is 0-2), S(O)n heteroaryl (where n is 0-2), S(O)n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and 5 combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents. 10 The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl" and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil. A "protected" moiety refers to a reactive functional group, e.g., a hydroxyl group or an amino group, or a class of molecules, e.g., sugars, having one or more functional groups, in which the reactivity of the functional group is temporarily blocked by the presence of an attached 15 protecting group. Protecting groups useful for the monomers and methods described herein can be found, e.g., in Greene, T.W., Protective Groups in Organic Synthesis (John Wiley and Sons: New York), 1981, which is hereby incorporated by reference. Structure of the Protected Monomers 20 In general, the protected monomer compounds include two differently functionalized hydroxyl groups (OFG' and OFG 2 below) and a ligand, all three of which are linked to a carrier molecule (see A below). As used herein, the term "functionalized hydroxyl group" means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFG 1 ) on the carrier is functionalized with a silicon 25 based protecting group. The other hydroxyl group (OFG 2 ) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. 48 WO 2004/094345 PCT/US2004/011822

OFG

1 OSiR 3 OSiR 3 carrier LIGAND carrier LIGAND carrier LIGAND

OFG

2 O LO~O N(R") 2 A B OR' C The above combination of substituents allows the monomers to be incorporated into an internal or terminal position of a natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomer compounds described herein and/or natural 5 or modifed ribonucleotides. The monomers described herein can therefore be used to prepare iRNA agents. While not wishing to be bound by theory, it is believed that incorporation of one or more of the monomers described herein can increase binding affinity of an iRNA agent to a target mRNA, increase nuclease resistence, change the geometry of the duplex form, alter distribution or target the iRNA agent to a particular part of the body, and modify the interaction 10 with nucleic acid binding proteins (e.g., during RISC formation and strand separation). When the OFG 2 in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed:to build up a chain of natural and/or modifed ribonucleotides once OFG' is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group 15 (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via the amidite group at OFG 2 . Subsequent to this operation, OFG 1 can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). 20 OFG 1 has the general formula D shown below. 49 WO 2004/094345 PCT/US2004/011822 X ' X -SiO- X D Hydroxyl groups, -OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a silicon-based protecting group, i.e. D, are essentially unreactive as nucleophiles 5 in displacement reactions. Thus, the silyl-protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. X5', X5", and X5'" can be selected from substituted or unsubstituted alkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy, cycloalkoxy, aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e., R 3 SiO-, the three "R" groups can be any combination of the above listed groups). X 5 ', X 5 ", and X 5 "' may all 10 be the same or different; also contemplated is a combination in which two of X s ', X 5 ", and X 5 ' are identical and the third is different. In certain embodiments X 5 ', X 5 ", and X 5 ' "' include at least one alkoxy or siloxy groups and may be any one of the groups listed in FIG. 2, a preferred combination includes X s ', X s

"

' = trimethylsiloxy and X 5 ' = 1, 3 -(triphenylmethoxy)-2-propoxy or cyclododecyloxy. 15 Other preferred combinations of X 5 ',

X

5 ' , and X s 5 ' include those that result in OFG 1 groups that meet the deprotection and stability criteria delineated below. The group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Rapid removal, i.e., less than one minute, of the silyl group from e.g., a support bound oligonucleotide is desirable because it can reduce synthesis times and thereby reduce 20 exposure timeof the growing oligonucleotide chain to the reagents. Oligonucleotide synthesis can be improved if the silyl protecting group is visible during deprotection, e.g., from the addition of a chromophore silyl substituent. Selection of silyl protecting groups can be complicated by the competing demands of the essential characteristics of stability and facile removal, and the need to balance these competitive 25 goals. Most substituents that increase stability can also increase the reaction time required for removal of the silyl group, potentially increasing the level of difficulty in removal of the group. The addition of alkoxy and siloxy substituents to OFG' increases the susceptibility of the protecting groups to fluoride cleavage of the silylether bonds. Increasing the steric bulk of the 50 WO 2004/094345 PCT/US2004/011822 substituents preserves stability while not decreasing fluoride lability to an equal extent. An appropriate balance of substituents on the silyl group makes a silyl ether a viable nucleoside protecting group. Candidate OFG 1 groups may be tested by exposing a tetrahydrofuran solution of a 5 preferred carrier bearing the candidate OFG 1 group to five molar equivalents of tetrahydrofuran at room temperature. The reaction time may be determined by monitoring the disappearance of the starting material by thin layer chromatography.

OFG

2 may have general formula E or F: Ok 1 or (R28) 2 N OR27 E F 10 OFG 2 in structure E is a phosphoramidite group and functions as a site where the monomer may be coupled to another monomer described herein or to a natural or modified ribonucleoside or oligoribonucleotide chain. For example, a ribonucleotide containing an unblocked 5'-OH can be coupled to the monomer via displacement of the N(R 28

)

2 moiety to form a phosphite ester linkage beween the monomer and the nucleoside. R 27 can be substituted or unsubstituted alkyl or 15 alkenyl. In preferred embodiments, R 27 is methyl, allyl or 2-cyanoethyl. R 28 may a C 1

-C

1 0 alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

OFG

2 in F is hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be 20 attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached 25 to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be -C(O)(CH 2 )qC(O)-, or -C(O)(CH 2 )qS-, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can 51 WO 2004/094345 PCT/US2004/011822 not be used in conjunction with fluoride labile 5' silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride stable polystyrene based supports or PEG are preferred. The carrier can be any organic molecule containing attachment points for OFG', OFG 2 , 5 and the ligand. In certain embodiments, carrier is a cyclic molecule and may contain heteroatoms (e.g., O, N or S). E.g., carrier molecules may include aryl (e.g., benzene, biphenyl, etc.), cycloalkyl (e.g., cyclohexane, cis or trans decalin, etc.), or heterocyclyl (piperazine, pyrrolidine, etc.). Any of the above cyclic systems may include substituents in addition to OFG 1,

OFG

2 , and the ligand. 10 In certain embodiments, the carrier is a nitogenous heterocycle. Exemplary carriers of this class include structures G and H. The designation "O" indicates possible locations for OFG' and OFG 2 . In certain embodiments of G, one of the piperazinyl nitrogens is substituted with hydrogen. In the case of structure H, the position left unoccupied by OFG' and OFG 2 can be substituted by a hydroxyl group, a protected hydroxyl group, or hydrogen. In both G and H, all 15 positional and stereoisomers are expressly contemplated. Preferred examples of H include H-p and H-ss in which "chol" represents a cholesterol radical (e.g., the oxygen attached to "chol" can be attached to C-3 of the cholesterol skeleton). The shaded circle in H-ss represents a liquid or solid support agent. 52 WO 2004/094345 PCT/US2004/011822 0 tether/linker-ligand O N N 0 0' 0 tether/linker-ligand G H 0 xs"-si-o 1 5,,, O-P-N(ipr)2 H-p I -- p OCH2CH2C N 0 X' ON O-chol 1 H x 5 ", H -C(O)CH 2

CH

2

C

(

0)NH H-ss 53 WO 2004/094345 PCT/US2004/011822 In other embodiments, the carrier molecule is an oxygen containing heterocycle. Preferably the carrier is a ribose sugar as shown in structure I. In this embodiment, the protected monomer is a nucleoside. X5' X5"-1Si-O B 0 15. x 3 X 2 I 5 "B" represents an "unusual" nucleobase or a "universal" base. As used herein, an "unusual" nucleobase can include any one of the following: 2-methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl, 10 N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl, N6-(cis-hydroxyisopentenyl)adeninyl, 2 -methylthio-N6-(cis-hydroxyisopentenyl) adeninyl, N6-glycinylcarbamoyladeninyl, 15 N6-threonylcarbamoyladeninyl, 2-methylthio-N6-threonyl carbamoyladeninyl, N6-methyl-N6-threonylcarbamoyladeninyl, N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl, 20 N6,N6-dimethyladeninyl, 3-methylcytosinyl, 5-methylcytosinyl, 2-thiocytosinyl, 5-formylcytosinyl, 25 54 WO 2004/094345 PCTIUS2004/01 1822 NH COOHN

H

2 N N N. H ~t N4-methylcytosinyl, 5 5-hydroxymethylc-ytosinyl, 1 -methylguaninyl, N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl, 10 55 WO 2004/094345 PCT/US2004/011822

NHCOOCH

3 NHCOOCH 3 NHCOOCH 3

H

3 COOC

H

3 COOC OH

H

3 COOC OOH 0 0 0 NN N N / N

H

3 C H C I

H

3 C

CH

3 CH 3 CH 3

NH

2 HOOC OH

H

3 C 0/N NN N 3C H3C

H

3 C / N N Na~ N> NNN~ N N N

CH

3 CH CHz O H H N2,7-dimethylguaninyl, 0 0 H0 H HN N H 3H N HOO HOH O t N' N N N 0 N 0 NH O NH N HN HN

H

2 N N N H 2 N N N HO HO beta-galactosylO O beta-mannosyl 0 O H NH 0 NH 0 NH 2 HN H, \ HN

H

2 N N N H 2 N N H 2 N N 5 56 WO 2004/094345 PCTIUS2004/01 1822 N2,N2,7-trimethylguaninyl, 1 -methylguaninyl, 7-cyano-.7-deazaguaninyl, 5 7-aminomethyl-7-deazaguaninyl, pseudouracilyl, dihydrouracilyl, 5-methyluracilyl, 1 -methyipseudouracilyl, i0 2-thiouracilyl, 4-thliouracilyl, 2-thiothyminyl 5-methyl-2-thiouracilyl,

-

3

-(

3 -amino-3-carboxypropyl)uracily, 15 5-hydroxyuracilyl, 5-methoxyuracilyl, uracilyl 5 -oxyacetic acid, uracilyl 5-oxyacetic acid methyl ester, 5-(carboxyhydroxymetliyl)uracilyl, 20 5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonyhmethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl, 5-aminomethyl-2-thiouracilyl, 5-methylaminomethyluracilyl, 25 5-methylaminomethyl-2-thiouracilyl, 5-methylamninomethyl-2-selenouracilyl, 5-carbamoylmethyluracilyl, 5-carboxymnethylaminomethyluracilyl, 5-carboxymnethylaminomethyl-2-thiouracilyl, 30 3-methyluracilyl, 1 -methyl-3 -( 3 -amino-3-carboxypropyl) pseudouracilyl, 5-carboxymethyluracilyl, 5-methyldihydrouracilyl, or 3-methyipseudouracilyl. 57 WO 2004/094345 PCT/US2004/011822 A universal base can form base pairs with each of the natural DNA/RNA bases, exhibiting relatively little discrimination between them. In general, the universal bases are non hydrogen bonding, hydrophobic, aromatic moieties which can stabilize e.g., duplex RNA or RNA-like molecules, via stacking interactions. A universal base can also include hydrogen 5 bonding substituents. As used herein, a "universal base" can include any one of the following: F CH 3

NH

2

NH

2 0 2 N

H

3 C N N N 'N ' I"> ' , F &N N0 2 0 0 BUHN N 0 N N N N 0 2 N : 0 H H 10 58 WO 2004/094345 PCT/US2004/011822

CH

3 0 CH 3 O O CH 3 N%- - H 3 C H 3 C N , HaC CH 3 O OH 3

H

3

CCHH

3 N N C ~

H

3 CCH3H 3 - ~- K- .- and / N

OH

3 A universal base can also include an aryl moiety (e.g., phenyl) having a ligand either directly attached or indirectly attached, e.g., via a linker or tether, to the aryl moiety. The aryl 5 moiety may further include additional substituents, e.g., one or more fluoro groups.

X

2 can include "oxy" or "deoxy" substituents in place of the 2'-OH. Examples of"oxy"-substituents include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, sugar, or protecting group); polyethyleneglycols (PEG),

O(CH

2

CH

2 0),CH 2

CH

2 OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, 10 e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-PROTECTED AMINE (AMINE = NH 2 ; alkylamnino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy,

O(CH

2 )nPROTECTED AMINE, (e.g., AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino), 15 and orthoester. Amine protecting groups can include formyl, amido, benzyl, allyl, etc. Preferred orthoesters have the general formula J. The groups R 31 and R 32 may be the same or different and can be any combination of the groups listed in FIG. 3. A preferred orthoester is the "ACE" group, shown below as structure K. 59 WO 2004/094345 PCT/US2004/011822 O

R

32 0

OR

3 1 J 0

H

3 C 0 O O

CH

3 O O K 0 "Deoxy" substituents include hydrogen (i.e. deoxyribose sugars, which are of particular 5 relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); protected amino (e.g.

NH

2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid in which all amino are protected); fully protected polyamino (e.g., NH(CH 2

CH

2 NH)nCH 2

CH

2 -AMINE, wherein AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino and all amino 10 groups are protected), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., a protected amino fimunctionality. Preferred substitutents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C- allyl, and 2'-fluoro.

X

3 is as described for OFG 2 above, and X 5 , Xs, and X 5 ' can be selected as discussed 15 above. In another embodiment, the carrier can be a carbocycle, or a sulfur-containing heterocycle. Synthesis and Use of the Protected Monomers A listing of ribonucleosides containing the unusual bases described herein are described 20 in "The RNA Modification Database" maintained by Pamela F. Crain, JefRozenski and James 60 WO 2004/094345 PCT/US2004/011822 A. McCloskey; Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, UT 84112, USA (RNAmods@lib.med.utah.edu) Carriers G and H can be synthesized by methods described herein and by those known in the art. 5 The protected nucleosides, e.g., compound I, can be employed in solid support synthesis of oligonucleotides. The 5' silyl protecting group can be used in conjunction with acid labile orthoesters at the 2' position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. Final deprotection conditions are known not to significantly degrade RNA products. Functional groups on the unusual and universal bases are blocked S10 during oligonucleotide synthesis with protecting groups that are compatible with the operations being performed that are described herein. All syntheses can be can be conducted in any automated or manual synthesizer on large, medium, or small scale. The syntheses may also be carried out in multiple well plates or glass slides. The 5'-O-silyl group can be removed via exposure to fluoride ions, which can include any 15 source of fluoride ion, e.g., those salts containing fluoride ion paired with inorganic counterions e.g., cesium fluoride and potassium fluoride or those salts containing fluoride ion paired with an organic counterion, e.g., a tetraalkylammonium fluoride. A crown ether catalyst can be utilized in combination with the inorganic fluoride in the deprotection reaction. Preferred fluoride ion source are tetrabutylammonium fluoride or aminehydrofluorides (e.g., combining aqueous [IF 20 with triethylamine in a dipolar aprotic solvent, e.g., dimethylformamide). The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters towards fluoride. Methyl protection of the phosphotriester or phosphitetriester can stabilize the linkage against fluoride ions and improve process yields. Since ribonucleosides have a reactive 2' hydroxyl substituent, it can be desirable to 25 protect the reactive 2' position in RNA with a protecting group that is compatible with a 5'-O silyl protecting group, e.g. one stable to fluoride. Orthoesters meet this criterion and can be readily removed in a final acid deprotection step that can result in minimal RNA degradation. Tetrazole catalysts can be used in the standard phosphoramidite coupling reaction. Preferred catalysts include e.g. tetrazole, S-ethyl-tetrazole, p-nitrophenyltetrazole. 30 The general process is as follows. Nucleosides are suitably protected and fmunctionalized for use in solid-phase or solution-phase synthesis of RNA oligonucleotides. The 2'-hydroxyl group in a ribonucleotide can be modified using a tris orthoester reagent. The 2'-hydroxyl can be modified to yield a 2'-O-orthoester nucleoside by reacting the ribonucleoside with the tris orthoester reagent in the presence of an acidic catalyst, e.g., pyridinium p-toluene sulfonate. 61 WO 2004/094345 PCT/US2004/011822 This reaction is known to those skilled in the art. The product can then be subjected to further protecting group reactions (e.g., 5'-O-silylation) and functionalizations (e.g., 3'-O phosphitylation) to produce a desired reagent (e.g., nucleoside phosphoramidite) for incorporation within an oligonucleotide or polymer by reactions known to those skilled in the art. 5 Preferred orthoesters include those comprising ethylene glycol ligands which are protected with acyl or ester protecting groups. Specifically, the preferred acyl group is acetyl. The nucleoside reagents may then be used by those skilled in the art to synthesize RNA oligonucleotides on commercially available synthesizer instruments, e.g. Gene Assembler Plus (Pharmacia), 380B (Applied Biosystems). Following synthesis (either solution-phase or solid 10 phase) of an oligonucleotide or polymer, the product can be subjected to one or more reactions using non-acidic reagents. One of these reactions may be strong basic conditions, for example, 40% methylamine in water for 10 minutes at 55.degree. C., which will remove the acyl protecting groups from the ethylene glycol ligands but leave the orthoester moiety attached. The resultant orthoester may be left attached when the polymer or oligonucleotide is used in 15 subsequent applications, or it may be removed in a final mildly-acidic reaction, for example, 10 minutes at 55.degree. C. in 50 mM acetic acid, pH 3.0, followed by addition of equal volume of 150 mM TRIS buffer for 10 minutes at 55.degree. C. The protected monomer compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or 20 recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on the bases) and protecting group methodologies (protection 25 and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., 30 Encyclopedia ofReagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. The protected monomer compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these 62 WO 2004/094345 PCT/US2004/011822 compounds are expressly included in the present invention. The compounds described herein can also contain linkages (e.g., carbon-carbon bonds, carbon-nitrogen bonds, e.g., amides) or substituents that can restrict bond rotation, e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans, E/Z isomers, and rotational isomers (rotamers) are 5 expressly included herein. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of 10 the compounds described herein are expressly included in the present invention. The monomers and methods described herein can be used to prepare natural or modified oligoribonucleotides, or polymeric molecules comprising any combination of monomer compounds described herein and/or natural or modified ribonucleotides in which one or more subunits contain an unusual or universal base. While not wishing to be bound by any theory, it is 15 believed that the incorporation of these bases can optimize binding affinity of an iRNA agent to a target mRNA, optimize endonuclease stability, increase the number of hydrogen bonding interactions, and create favorable pi-stacking, polarizability and sugar pucker in the duplex form. Unusual and universal bases can be incorporated into both the sense and anti-sense strands of an iRNA agent. In preferred embodiments, the monomers and methods described herein can be 20 used to introduce a unusual or universal base-containing subunit into the 3' terminal position of the natural oligoribonucleotide, modified oligoribonucleotide, or polymer, and/or the 5' terminal position of natural oligoribonucleotide or modified oligoribonucleotide or polymer. Universal bases are described in "Survey and Summary: The Applications of Universal DNA base analogues" Loakes, D., Nucleic Acid Research 2001, 29, 2437, which is incorporated 25 by reference in its entirety. Specific examples are described in the following: Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926; Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632; Matray, T, J.; Kool, E. T. J Am. Chem. Soc., 1998, 120, 6191-6192; Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J Am. Chem. Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.; 30 Ogawa, A. K.; McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J.1 Am. Chem. Soc., 2000, 122, 3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804; Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440; Wu, Y.; Ogawa, A. 63 WO 2004/094345 PCT/US2004/011822 K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J Am. Chem. Soc., 2000, 122, 7621-7632;. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.; Tor, Y. J Am. Chem. Soc., 2001, 123, 3375 5 3376; Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13. In certain embodiments the methods and monomers described herein can be used to prepare pseudocomplementary double-stranded iRNA agents that contain one or more interstrand pairings between unusual bases, e.g., 2-aminoadenine (2-AA) and 2-thiouracil (2-TU). A monomer of general structure I having a 2-amino adenine nucleobase can be incorporated into 10 first strand and a monomer of general structure I having a 2-thiouracil nucleobase can be incorporated into a second strand. While not wishing to be bound by any theory, it is believed that the ground state of the resultant duplex containing the 2-AA - 2-TU pairing will be destabilized relative to the ground state of a duplex containing a 2-AA - uracil or 2-TU adenine pairing. Again, while not wishing to be bound by any theory, it is believed that this 15 ground state destabilization can facilitate helicase activity, which is involved in strand separation of the duplex during processing. 2-aminoadenine and 2-thiouracil-containing oligonucletide strands are described in "Oligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agents." Kutyavin, Igor V.; Rhinehart, Rebecca L.; Lukhtanov, Eugeny A.; Gorm, Vladimir V.; Meyer, Rich B., Jr.; Gamper, Howard B., Jr. Epoch 20 Pharmaceuticals Inc., Bothell, WA, USA. Biochemistry (1996), 35(34), 11170-11176; and "Double duplex invasion by peptide nucleic acid: a general principle for sequence-specific targeting of double-stranded DNA." Lohse, Jesper; Dahl, Otto; Nielsen, Peter E.. Center for Biomolecular Recognition, Department of Chemistry, University of Copenhagen, Copenhagen, Den. Proceedings of the National Academy of Sciences of the United States ofAmerica (1999), 25 96(21), 11804-11808. As discussed above, the monomers and methods described herein can be used in the preparation of modified RNA molecules. Modified RNA molecules include e.g. those molecules containing a chemically or stereochemrnically modified nucleoside or a nucleoside surrogate. Coupling of 5'-hydroxyl groups with phosphoramidites forms phosphite ester intermediates, 30 which in turn are oxidized e.g., with iodine, to the phosphate diester. Alternatively, the phosphites may be treated with e.g., sulfur, selenium, amino, and boron reagents to form modified phosphate backbones. Linkages between the monomers described herein and a nucleoside or oligonucleotide chain can also be treated with iodine, sulfur, selenium, amino, and boron reagents to form unmodified and modified phosphate backbones respectively. Similarly, 64 WO 2004/094345 PCT/US2004/011822 the monomers described herein may be coupled with nucleosides or oligonucleotides containing any of the modifications or nucleoside surrogates described herein. Ligands 5 The monomers of the invention can be derivatized with a ligand, as opposed to a base. Preferred ligands are moieties, other than naturally occuring bases (A, T, G, C, and U), that are coupled, preferrably covalently, to the sugar or carrier moiety of the monomer. In preferred embodiments a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected 10 target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species derivatized with one of the bases A, G, T, C, or U. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid. Preferred ligands can improve transport, hybridization, and specificity properties and may also improve improve nuclease resistance of the resultant natural or modified 15 oligoribonucleotide, or a polymeric molecule comprising any combination of monomer compounds described herein and/or natural or modifed ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples 20 include lipids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic 25 polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2 hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or 30 polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide. 65 WO 2004/094345 PCT/US2004/011822 Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent 5 galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers 10 (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1, 3 -Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03 15 (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, 20 imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP. Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specfic affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone 25 receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharid, an activator of p38 MAP kinase, or an activator of NF-KB. The ligand can be a substance, e.g, a drug, that can increase the uptake of the iRNA agent 30 into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. 66 WO 2004/094345 PCT/US2004/011822 The ligand, e.g., when a drug can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based 5 molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the 10 conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likey to be cleared from the body. A 15 lipid or lipid-based ligand that binds to USA less strongly can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding 20 cannot be reversed. In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand. In a preferred embodiment, the lipid or lipid based ligand is a phosphorothioate. In this 25 embodiment, it is preferred that the number of sulfurs on the phosphorothioate not be so prevalent that they interfere with binding to a serum protein, e.g., HSA. In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. 30 Exemplary vitamins are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL). In another aspect, the ligand is a cell-permeation agent, preferably a helical cell permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as 67 WO 2004/094345 PCT/US2004/011822 tat or antennopedia. If the agent is a peptide, it can be modified, including a pepidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. 5 Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and petomimetics can target cancer cells, in particular cells that exhibit an aI3 3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the av- 3 3 integrin ligand. Generally, such ligands can be used 10 to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein. The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of a monomore derivitzed with a ligand which targets to the liver. For example, a liver-targeting agent can be a lipophilic moiety. 15 Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues (see Table 1). Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3 propanediol, heptadecyl group, palmnitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03 20 (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver. A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is 25 particularly useful. These agents target, in particular, the parenchymal cells of the liver (see Table 1). For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or 30 mannose-6-phosphate targeting moiety can be used for macrophage targeting. Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver. 68 WO 2004/094345 PCT/US2004/011822 An iRNA agent can be targeted to a particular cell type in the liver by using specific targeting agents, which recognize particular receptors in the liver. Exemplary targeting moieties and their associated receptors are presented in Table 1. 5 Table 1 Targeting agents (Ligands) and their associated receptors Liver Cells Ligand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC) (Hepatocytes) (Asiologlycoprotein receptor) Gal NAc ASPG-R (n-acetyl- Gal NAc Receptor galactosamine) Lactose Asialofetuin ASPG-r 2) Sinusoidal Hyaluronan Hyaluronan receptor Endothelial Cell (SEC) Procollagen Procollagen receptor Negatively charged Scavenger receptors molecules Mannose Mannose receptors N-acetyl Scavenger receptors Glucosamine Immunoglobulins Fe Receptor LPS CD14 Receptor Insulin Receptor mediated transcytosis Transferrin Receptor mediated transcytosis Albumins Non-specific Sugar-Albumin conjugates Mannose-6- Mannose-6-phosphate phosphate receptor 69 WO 2004/094345 PCT/US2004/011822 3) Kupffer Cell Mannose Mannose receptors (KC) Fucose Fucose receptors Albumins Non-specific Mannose-albumin conjugates The selection of ligands is within skill of the art, and useful candidate ligands can be identified by routine methods. Ligands can be can be connected to the carrier via a tether, which may be selected from 5 C(O)-(CH 2 )s-C(O)-(ligand); -C(O)-(CH 2 )s-C(O)O-(ligand); -C(O)-O-(ligand); -C(O)-(CH2)s-NH-; -C(O)-(CH2)s-NH-C(O)-(ligand); -C(O)-(CH2)s-(ligand); -C(O)-NH-(ligand); -C(O)-(ligand); -(CH2)s-C(O)-(ligand); -(CH2)s-C(0)O-(ligand); -(CH2)s-(ligand); -(CH2)s-NH-; or -(CH 2 )s-NH-C(O)-(ligand); s can be 0-20, preferably 0-4. 10 iRNA AGENT STRUCTURE The monomers described herein can be used to make oligonucleotides which are useful as iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi, e.g., with respect to an endogenous gene of a subject or to a gene of a pathogen. In most cases the iRNA agent will incorporate momomers described herein together with naturally occuring 15 nucleosides or nucleotides or with other modified nucleosides or nucleotides. The modified monomers can be present at any position in the iRNA agent, e.g., at the terminii or in the middle region of an iRNA agent or in a duplex region or in an unpaired region. In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, a hairpin or other single 20 strand structure or a two-strand structure, as described herein. An "RNA agent" as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. 25 Preferred examples include those which have a 2' sugar modification, a modification in a single strand overhang, preferably a 3' single strand overhang, or, particularly if single stranded, a 5' 70 WO 2004/094345 PCT/US2004/011822 modification which includes one or more phosphate groups or one or more analogs of a phosphate group. An "iRNA agent" as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an 5 endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is 10 particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group. The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or 15 ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term "ribonucleotide" or "nucleotide", herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, 20 complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. Complementarity, or degree of homology with the target strand, is most critical in the 25 antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3' 30 terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule. 71 WO 2004/094345 PCT/US2004/011822 As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the ribose replacement modification subunit (RRMS). Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links 5 two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5'-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that 10 come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis. iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not 15 trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. "sRNA agent or shorter iRNA agent" as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent 20 or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA. 25 Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3' overhangs, of 2-3 nucleotides. 30 In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties: (1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below; 72 WO 2004/094345 PCT/US2004/011822 (2) if single stranded it will have a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group; (3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper 5 three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA; (4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have "RNA-like" properties, i.e., it will possess the overall structural, chemical 10 and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an 15 axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C 3 ,-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. 20 (Generally, it is preferred that a modified moiety at the 2' sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C 3 -endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a C 3 '-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more 25 than 20, 10, 5, 4, 3, 2, orl sugar which is not a C 3 ,-endo pucker structure. These limitations are particularly preferably in the antisense strand; (5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 30 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent. 73 WO 2004/094345 PCT/US2004/011822 A "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA 5 agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5' phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O 10 P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha 15 thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether-methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). (These modifications can also be used with the antisense strand ofa double stranded iRNA.) 20 A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length. Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 25 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length. 30 A "double stranded (ds) iRNA agent" as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure. 74 WO 2004/094345 PCT/US2004/011822 The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length. 5 The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length. The double strand portion of a double stranded iRNA agent should be equal to or at least, 10 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents 15 It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to 20 inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5'-phosphorylation of the sense strand, e.g., by modification with a 5'-O-methyl ribonucleotide (see Nykqnen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting 25 the 5'-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5' phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5'-end. Antisense strand modifications include 5' phosphorylation as well as any of the other 5' modifications discussed herein, particularly the 5' modifications discussed above in the section on single 30 stranded iRNA molecules. 75 WO 2004/094345 PCT/US2004/011822 It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments 5 will have a 3' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5' ends are preferably phosphorylated. Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 10 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3' overhang are also within the invention. 15 The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, 20 can also be targeted. As used herein, the phrase "mediates RNAi" refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides. 25 As used herein, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., 30 under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides. 76 WO 2004/094345 PCT/US2004/011822 In one embodiment, an iRNA agent is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is "exactly complementary" (excluding the RRMS containing subunit(s))to a target RNA, e.g., the target RNA and the iRNA agent 5 anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in 10 the region (e.g., within 7 nucleotides of) the single-nucleotide difference. As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides. RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. 15 Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual 20 RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred 25 to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of 30 all of the above are discussed herein. Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is 77 WO 2004/094345 PCT/US2004/011822 required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described 5 elsewhere herein. As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and 10 infact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a 15 phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5' end or ends can be phosphorylated. In some embodiments it is particularly preferred, e.g., to enhance stability, to include 20 particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3' or 5' overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of 25 deoxyribonucleotides, e.g., deoxythymidine, instead ofribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence. 78 WO 2004/094345 PCT/US2004/011822 Modifications and nucleotide surrogates are discussed below. II 5BS fll w " OH 2 H BASE 0 SO H (2' OH) X-- Y ~I z I BASE 0 3 2OH (2'OH) FORMULA 1 5 79 WO 2004/094345 PCT/US2004/011822 The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2' hydroxyl ribose sugar (as depicted) and W, 5 X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide. Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications o10 to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent. Modified nucleic acids and nucleotide surrogates can include one or more of: 15 (i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5' end of a nucleic acid and the 20 terminal Z position at the 3' end of a nucleic acid, are within the term "linking phosphate oxygens" as used herein.); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein; 25 (iii) wholesale replacement of the phosphate moiety (bracket I) with "dephospho" linkers; (iv) modification or replacement of a naturally occurring base; (v) replacement or modification of the ribose-phosphate backbone (bracket II); 80 WO 2004/094345 PCT/US2004/011822 (vi) modification of the 3' end or 5' end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3' or 5' end of RNA. The terms replacement, modification, alteration, and the like, as used in this context, do 5 not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule. It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to 10 be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or "localization" of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a 15 greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X = O and Y = N in the above figure. 20 Specific modifications are discussed in more detail below. The Phosphate Group The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. 25 One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, 30 borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl 81 WO 2004/094345 PCT/US2004/011822 or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which 5 the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, 10 or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred. The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen 15is (position W (3') or position Z (5'). Replacement of W with carbon or Z with nitrogen is preferred. Candidate agents can be evaluated for suitability as described below. The Sugar Group A modified RNA can include modification of all or some of the sugar groups of the 20 ribonucleic acid. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments 25 to introduce alterations in which alkoxide formation at the 2' position is not possible. Examples of"oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),

O(CH

2

CH

2 0)nCH 2

CH

2 OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE = 30 NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or 82 WO 2004/094345 PCT/US2004/011822 diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH 2 )nAMINE, (e.g., AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH 2

CH

2

OCH

3 , a PEG 5 derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification. "Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl 10 amino, or amino acid); NH(CH 2

CH

2 NH)nCH 2

CH

2 -AMINE (AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2'-methoxyethyl, 2' 15 OCH3, 2'-O-allyl, 2'-C- allyl, and 2'-fluoro. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar. Modified RNAs can also include "abasic" sugars, which lack a nucleobase at C-I'. These 20 abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms. To maximize nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric" oligonucleotides are those that contain two or more different modifications. 25 The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs. Candidate modifications can be evaluated as described below. 83 WO 2004/094345 PCT/US2004/011822 Replacement of the Phosphate Group The phosphate group can be replaced by non-phosphorus containing connectors (cf Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its 5 replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety. Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, 10 thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups. Candidate modifications can be evaluated as described below. Replacement of Ribophosphate Backbone 15 Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable 20 in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone. Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate. Candidate modifications can be evaluated as described below. 25 Terminal Modifications The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3' 84 WO 2004/094345 PCT/US2004/011822 and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal 5 atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., (CH 2 )n-, -(CH 2 )nN-, -(CH 2 )nO-, -(CH 2 )nS-, O(CH 2

CH

2 0)nCH 2

CH

2 OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, 10 or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3' end can be an OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not 15 wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic 20 acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, 25 substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles). Terminal modifications can be added for a number of reasons, including as discussed 30 elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate 85 WO 2004/094345 PCT/US2004/011822 modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5' 5 adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5' (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HIS)(S)P-O-5'), 5' phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma o10 thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NHI2)(O)P-O-5'), 5' alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). Terminal modifications can also be useful for monitoring distribution, and in such cases 15 the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross linking an RNA agent to another moiety; modifications useful for this include mitomycin C. Candidate modifications can be evaluated as described below. 20 The Bases Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or 25 tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., "unusual bases" and "universal bases" described herein, can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and 30 thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5 86 WO 2004/094345 PCT/US2004/011822 substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5 azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 5 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2 thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3 methylcytosine, 5-methylcytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6 isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. 10 Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Generally, base changes are less preferred for promoting stability, but they can be useful 15 for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents. Candidate modifications can be evaluated as described below. Evaluation of Candidate RNA's 20 One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, 25 which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive 30 or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control 87 WO 2004/094345 PCT/US2004/011822 molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule. 5 A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA 10 agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on 15 gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents. In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in 20 vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA 25 levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non modified RNA is added. 88 WO 2004/094345 PCT/US2004/011822 References General References The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example "Oligonucleotide synthesis, a practical 5 approach", Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A Practical Approach", Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modem machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-O--Methyloligoribonucleotide- s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, 10 Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223 2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references 15 referred to therein. Modification described in WO 00/44895, WO01/75164, or W002/44321 can be used herein. The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference. 20 Phosphate Group References The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester 25 oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3'-Deoxy-3'-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described in An, H, et al. J Org. Chemin. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described 89 WO 2004/094345 PCT/US2004/011822 in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693. Sugar Group References Modifications to the 2' modifications can be found in Verma, S. et al. Annu. Rev. 5 Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831 841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), "LNA" (Wengel, J. Acc. Chemn. Res. 1999, 32, 301-310). Replacement of the Phosphate Group References 10 Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked 15 oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/UJS92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 20 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier,J.F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J.R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M.D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E.P. 25 Nucleic Acids Res. 1989, 17, 6129. Replacement of the Phosphate-Ribose Backbone References Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 30 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are 90 WO 2004/094345 PCT/US2004/011822 ' known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083. 5 Terminal Modification References Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein. Bases References N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat. 10 No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications. 15 Preferred iRNA Agents Preferred RNA agents have the following structure (see Formula 2 below): 91 WO 2004/094345 PCT/US2004/011822 Al 0 R, R7 rx

A

2 R4 0 R2 R7 .s ;

A

3

R

5

R

3 R7 A6 FORMULA 2 Referring to Formula 2 above, R', R 2 , and R 3 are each, independently, H, (i.e. abasic 5 nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, 10 amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5 trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5 azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7 15 deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3 92 WO 2004/094345 PCT/US2004/011822 methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5 methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino 3carboxypropyl)uracil, 3-methyleytosine, 5-methyleytosine, N 4 -acetyl cytosine, 2-thiocytosine, 5 N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N methylguanines, or O-alkylated bases.

R

4 , R, and R 6 are each, independently, OR , O(CH 2

CH

2 0)mCH 2 CH2OR ; O(CH 2 )nR 9 ;

O(CH

2 )nOR 9 , H; halo; NH 2 ; NIHR; N(R 8

)

2 ; NH(CH 2

CH

2 NII)mCH 2

CH

2

NHR

9 ; NHC(O)Rs; ; cyano; mercapto, SR ; alkyl-thio-alkyl; allkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, 10 alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or 15 ureido; or R 4 , R s , or R 6 together combine with R 7 to form an [-O-CH 2 -] covalently bound bridge between the sugar 2' and 4' carbons. 93 WO 2004/094345 PCT/US2004/011822 A' is: Wi Xl -- p~y 1 X4= P-Y 1 I o, I W, Z, WI I Xl=P- Yi or X1P- Y, W, or i x 1 =P-Y_ 1-1 X,-P- Y1 XI= P- Y1 I I I Zi V 5 ; H; OH; OCH 3 ; W 1 ; an abasic nucleotide; or absent; (a preferred Al , especially with regard to anti-sense strands, is chosen from 5' monophosphate ((HO) 2 (O)P-O-5'), 5'-diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'), 5' triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-adenosine cap 10 (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O (HO)(O)P-O-P(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P-O-5'), 5' monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate

((HO)

2 (O)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 15 5'-phosphoramidates ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH) 2 (O)P-5'-CH 2 -), 5' alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH 2 -), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-)).

A

2 is: 94 WO 2004/094345 PCT/US2004/011822

X

2 -P-Y2 12

A

3 is:

X

3 = P Y 3 Z3 ; and 595 95 WO 2004/094345 PCT/US2004/011822

A

4 is: I

X

4 I I X 4 -P- Y 4 or X4 P- Y4 or i

X

4 P Y 4 X4 P Y4 X44 Z4 I I

Z

4

Z

4

Z

4 5 ;H; Z 4 ; an inverted nucleotide; an abasic nucleotide; or absent. W' is OH, (CH 2 )nR' 0 , (CH 2 )nNHR' 10 , (CH 2 )n ORo ,

(CH

2 )n SR10I; O(CH 2 )nRI;

O(CH

2 )nOR 1 0 , O(CH 2 )nNR' 0 , O(CH 2 )nSRio; O(CH 2 )nSS(CH 2 )nORio, O(CH 2 )nC(O)OR 1 0 ,

NH(CH

2 )nRIo; NH(CH 2 )nNR 1 0

;NH(CH

2 )nOR 1 0 , NH(CH 2 )nSR'o; S(CH 2 )nRi 0 , S(CH 2 )nNR" 1 ,

S(CH

2 )nOR'o, S(CH 2 )nSR'o O(CH 2

CH

2 0)mCH 2

CH

2 ORi; O(CH 2

CH

2 0)mCH 2

CH

2 NHR1 0 , 10 NH(CH 2

CH

2 NH)mCH 2

CH

2

NHRI

0 ; Q-Rio, O-Q-R'o N-Q-R 1 o, S-Q-R 1 o or -0-. W 4 is O, CH 2 , NH, or S. X', X 2 , X 3 , and X 4 are each, independently, O or S.

Y

1 , Y 2 , Y 3 , and Y 4 are each, independently, OH, O0, OR', S, Se, BH3-, H, NHR 9 , N(R 9

)

2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted. 15 Z', Z 2 , and Z 3 are each independently O, CH 2 , NH, or S. Z 4 is OH, (CH 2

),R

1 0 ,

(CH

2 )nNHIR' 0 , (CH 2 )n ORo ,

(CH

2 )n SRIo; O(CH 2 )nR'I 0 ; O(CH 2 )nOR'o, O(CH 2 )nNR',

O(CH

2 )nSRo, O(CH 2 )nSS(CH 2 )nOR'o, O(CH 2 )nC(O)ORio; NIHI(CH 2 )nRi 0 ; NH(CH 2 )nNR' 0

;NH(CH

2 )nOR'o, NH(CH 2 )nSR'Io; S(CH 2 )nR 1 o, S(CH 2 )nNR 1 0 , S(CH 2 )nOR'o, S(CH 2 )nSR 1 0 96 WO 2004/094345 PCT/US2004/011822

O(CH

2

CH

2 0)mCH 2

CH

2

OR

1 0 , O(CH 2

CH

2 0)mCH 2

CH

2 NHR 10 ,

NH(CH

2

CH

2 NH)mCH 2

CH

2 NHRIo; Q-R'o, O-Q-R 10 N-Q-Rio ,

S-Q-R

1 0 . x is 5-100, chosen to comply with a length for an RNA agent described herein.

R

7 is H; or is together combined with R 4 , R 5 , or R 6 to form an [-O-CH 2 -] covalently 5 bound bridge between the sugar 2' and 4' carbons.

R

8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R 9 is

NH

2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R10 is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), 10 cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic 15 acid,03-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, 20 histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents. Preferred RNA agents in which the entire phosphate group has been replaced have the 25 following structure (see Formula 3 below): 97 WO 2004/094345 PCT/US2004/011822 Alo R1o 0

R

70 Ao R40 R20 0

R

70 AR50

R

30 0

R

70

A

4 0 R 60 o FORMULA 3 5 Referring to Formula 3, Alo-A40 is L-G-L; A"o and/or A 40 may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH 2

(CH

2 )g; N(CH2)g; O(CH2)g; S(CH 2 )g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, 10 methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R

10 , R 20 , and R 30 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, 98 WO 2004/094345 PCT/US2004/011822 isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5 halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, 5 hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5 substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7 alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6 10 diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5 methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5 methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5 methylcytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2 15 methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R

40 , R 5 o, and R 6 o are each, independently, OR 8 , O(CH 2

CH

2 0)mCH 2

CH

2 OR; O(CH 2 )nR';

O(CH

2 )nOR 9 , H; halo; NH 2 ; NIRS; N(R) 2 ; NH(CH 2

CH

2 NH)mCH 2

CH

2

R

9 ; NHC(O)R;; cyano; mercapto, SR 7 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, 20 aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R 40 ,

R

5 0 , or R 6 o together combine with R 7 0 to form an [-O-CH 2 -] covalently bound bridge between the 25 sugar 2' and 4' carbons. x is 5-100 or chosen to comply with a length for an RNA agent described herein.

R

7 is H; or is together combined with R 4 0 , R 5 o, or R 6 0 to form an [-O-CH 2 -] covalently bound bridge between the sugar 2' and 4' carbons.

R

8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and 30 R 9 is NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2. 99 WO 2004/094345 PCT/US2004/011822 Preferred nucleoside surrogates have the following structure (see Formula 4 below):

SLR

00

-(M-SLR

2 oo)x-M-SLR 3 00 FORMULA 4 5 S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH2(CH2)g; N(CH2)g; O(CH2)g; S(CH 2 )g; -C(O)(CH 2 )n-or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may o10 be absent.

R

1 ° c, R200, and R300 are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl 15 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5 halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5 substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil 20 and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7 alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6 diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1, 2, 4,-triazoles, 2 pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5 methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5 25 methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5 methylcytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2 methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2. 100 WO 2004/094345 PCT/US2004/011822 Nuclease resistant monomers The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, that also incorporates a nuclease resistant monomer (NRM), such as those described herein and those described in copending, co-owned United States Provisional Application Serial 5 No. 60/469,612, filed on May 9, 2003, and International Application No. PCT/US04/07070, both of which are hereby incorporated by reference. An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or nuclease resistance promoting 10 monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule. 15 While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the half-life of the iRNA agent prior 20 to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3' and/or 5' end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that 25 placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting. Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the 30 sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3' end and/or the 5' end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a 101 WO 2004/094345 PCT/US2004/011822 duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3' end and/or the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular 5 configurations are discussed below. Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone: (i) chiral (Sp) thioates. Thus, preferred NRMs include nucleotide dimers with an enriched 10 or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr 2 , or Br 3 . When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRMs are discussed in more detail below; 15 (ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRMs include monomers at the terminal position derivatized at a cationic group. As the 5' end of an antisense sequence should have a terminal -OH or phosphate group this NRM is preferably not used at the 5' end of an anti-sense sequence. The group should be attached at a position on the base which 20 minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g, at the 5' position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below; (iii) nonphosphate linkages at the termini. Thus, preferred NRMs include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a 25 phosphate bond. Examples include 3' CH2-NCH 3 -O-CH2-5' and 3' CH2-NH-(O=)-CH2-5'.; (iv) 3'-bridging thiophosphates and 5'-bridging thiophosphates. Thus, preferred NRM's can included these structures; (v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2'-5' 30 phosphate, non-phosphate and modified phosphate linkages (e.g., thiophosphates, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3'-3' or 5'-5' linkages; monomers having an alpha linkage at the 1' site on the sugar, e.g., the structures described herein having an alpha linkage; 102 WO 2004/094345 PCT/US2004/011822 (vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone; (vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an 5 abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and (vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, 10 are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an unprotected phosphate. 15 One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintained. 20 In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing. Chiral Sp Thioates 25 A modification can include the alteration, e.g., replacement, of one or both of the non linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moieties, SBI and SB 2 . 103 WO 2004/094345 PCT/US2004/011822 SBI I Z_

SB

2 5 FORMULA X In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR 2 (R is hydrogen, 10 alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). 15 Thus if 60% of a population of stereogenic phosphorus atoms have the Rp configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the Sp configuration. In some embodiments, iRNA agents, having phosphate groups in which a phosphate non linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 20 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Sp configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least 25 about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Rp configuration. In other embodiments, the population of stereogenic phosphorus atoms may have 104 WO 2004/094345 PCT/US2004/011822 the Sp configuration and may be substantially free of stereogenic phosphorus atoms having the Rp configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the Sp configuration. As used herein, the phrase "substantially free of stereogenic 5 phosphorus atoms having the Rp configuration" means that moieties containing stereogenic phosphorus atoms having the Rp configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the Sp configuration"' means that moieties containing stereogenic phosphorus atoms having the Sp 10 configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.). In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic 15 phosphorus atoms may have the Sp configuration and be substantially free of stereogenic phosphorus atoms having the Rp configuration. Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X 1 and X-2. The former can be used to introduce phosphorothioate 105 WO 2004/094345 PCT/US2004/011822 DMTO DMTO O BASE O BASE SR2 W R2' I I S=P-Y S=P-Y Z Z O BASE O BASE O R2' O R21 I NC I solid phase reagent P 0 N(ipr) 2 X-1 X-2 at the 3' end of a strand, while the latter can be used to introduce this modification at the 5' end 5 or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R 2 . can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH 3 ), DMT is dimethoxytrityl, and "BASE" can be a natural, unusual, or a universal base. X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in 10 phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the Rp configuration (i.e., being substantially free of the Sp configuration) or only the Sp configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the Rp configuration and about 50% of the atoms have the Sp 15 configuration. Dimers having stereogenic phosphorus atoms with the Rp configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the Sp configuration using e.g., enzymatic degradation and/or conventional chromatography techniques. 106 WO 2004/094345 PCT/US2004/011822 Cationic Groups Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or 5 universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2' position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl 10 o amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH 2 )nAMIN E, (e.g., AMINE = NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH 2

CH

2 NH)nCH 2

CH

2 -AMINE (AMINE = NH 2 ; 15 alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino). Nonphosphate Linkages Modifications can also include the incorporation ofnonphosphate linkages at the 5' and/or 3' end of a strand. Examples of nonphosphate linkages which can replace the phosphate 20 group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups. 25 3'-bridging thiophosphates and 5'-bridging thiophosphates; locked-RNA, 2'-5' likages, inverted linkages, ca-nucleosides; conjugate groups; abasic linkages; and 5'-phosphonates and 5'-phosphate prodrugs Referring to formula X above, modifications can include replacement of one of the 30 bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom. 107 WO 2004/094345 PCT/US2004/011822 Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2' position of a first sugar and the 5' position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3' positions. Modified RNA's can also include "abasic" sugars, which 5 lack a nucleobase at C-I'. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the cc-configuration. Modifcations can also include L-RNA. 10 Modifications can also include 5'-phosphonates, e.g., P(O)(CT) 2

-X-C

5 '-sugar (X= CH2, CF2, CHF and 5'-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R] 2 CH2 C 5 '-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular SN2 displacement can depart as episulfide to afford the underivatized phosphate group. 15 Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation. Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit 20 hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene.. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a 25 sequence which does not target a subject sequence or gene,as it can minimize off-target silencing. In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent-it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the 30 iRNA agent. In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which 108 WO 2004/094345 PCT/US2004/011822 interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides 5 upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.) Such modifications can be introduced into the terminal regions, e.g., at the terminal 10 position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject. An iRNA agent can have a first and a second strand chosen from the following: a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end; 15 a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end; a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end; 20 a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region; a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 25 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3' and the 5' end; and a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end; a second strand which targets a sequence and which has an NRM modification at or 30 within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand); 109 WO 2004/094345 PCT/US2004/011822 a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end; a second strand which targets a sequence and which preferably does not have an an NRM 5 modification at the cleavage site or in the cleavage region; a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions 10 from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand). An iRNA agent can also target two sequences and can have a first and second strand chosen from: a first strand which targets a sequence and which has an NRM modification at or within 15 1, 2, 3, 4, 5, or 6 positions from the 3' end; a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand); 20 a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end and which has a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end; a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region; 25 a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, orNRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but 30 rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand) and a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end; a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially 110 WO 2004/094345 PCT/US2004/011822 not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand); a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end and which has a NRM modification at or 5 within 1, 2, 3, 4, 5 , or 6 positions from the 5' end; a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region; a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or 10 within 1, 2, 3, 4, 5, or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand). 15 Ribose Mimics The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, that also incorporates a ribose mimic, such as those described herein and those described in copending co-owned United States Provisional Application Serial No. 60/454,962, filed on March 13, 2003, and International Application No. PCT/USO4/07070, both of which are 20 hereby incorporated by reference. Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers 25 nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3' ribose hydroxyl group, thereby causing it to be less susceptible to degradation. 30 The secondary hydroxyl group refers to an "OH" radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the 111 WO 2004/094345 PCT/US2004/011822 terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base.. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal 5 phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group. Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see 10 formula Y below):

A

W I Y-P=--X z

CH

2 R3 \R4

R

1

CC

H NHT

R

2 R5

OR

7 R6 (Y) 15 Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to "W" of the phosphate group. These spacers or linkers can include e.g., -(CH 2 )n-, -(CH 2 )nN-, (CH 2 )nO-, -(CH 2 )nS-, O(CH 2

CH

2 0)nCH 2

CH

2 OH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin 20 and fluorescein reagents. The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH 3 -, C1-C 6 alkyl, C 6 -Co 10 aryl, H, O, O-, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S. 112 WO 2004/094345 PCT/US2004/011822 R, and R 3 are each, independently, hydrogen; or C 1 -Coo 00 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R

2 is hydrogen; C 1 -CIo 00 alkyl, optionally substituted with hydroxyl, amino, halo, 5 phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 2 may be taken together with with R 4 or R 6 to form a ring of 5-12 atoms.

R

4 is hydrogen; C 1

-C

1 00 oo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 4 may be taken together with with R 2 or R5 to form a ring of 5-12 atoms. 10 R 5 is hydrogen, C 1 -Cloo alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 5 may be taken together with with R 4 to form a ring of 5-12 atoms.

R

6 is hydrogen, C 1 -Co 10 o alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when 15 n is 1, R 6 may be taken together with with R 2 to form a ring of 6-10 atoms; R7 is hydrogen, C 1 -Co 100 alkyl, or C(O)(CH 2 )qC(O)NHR 9 ; T is hydrogen or a functional group; n and q are each independently 1-100; Rs is C 1 -Co 10 alkyl or C 6

-C

1 0 aryl; and R9 is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent. Preferred embodiments may include one of more of the following subsets of iRNA agent 20 delivery modules. In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3' or 5' ribose sugar carbon of the RNA agent. In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S. In still yet another subset of RNAi agent delivery modules, n is 1, and R 2 and R 6 are 25 taken together to form a ring containing six atoms and R 4 and R5 are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of 113 WO 2004/094345 PCT/US2004/011822 Formula (Y-l): A INHT HO 5 The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), 10 multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin. As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.The synthesized compounds can be separated from a reaction mixture and further purified by a method such as 15 column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, 20 Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.GM. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia ofReagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. 25 Ribose Replacement Monomer Subunits iRNA agents can be modified in a number of ways which can optimize, one or more characteristics of the iRNA agent. The monomers and methods described herein can be used to 114 WO 2004/094345 PCT/US2004/011822 prepare an RNA agent, e.g., an iRNA agent, that includes a ribose replacement monomer subunit (RRMS), such as those described herein and those described in one or more of United States Provisional Application Serial No. 60/493,986, filed on August 8, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/494,597, filed on 5 August 11, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/506,341, filed on September 26, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/158,453, filed on November 7, 2003, which is hereby incorporated by reference; and International Application No. PCT/US04/07070, filed March 8, 2004, which is hereby incorporated by reference. The 10 synthetic methods and modifications described in these application can be used with or combined with the monomers and methods described herein. In addition, the monomers and methods described herein can be used to prepare iRNA agents having an RRMS and another element described herein. E.g.,. The monomers and methods described herein can be used to prepare an iRNA agent described herein, e.g., a 15 palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the kidney, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, 20 which also incorporates a RRMS. The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a 25 carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds. The carriers further include (i) at least two "backbone attachment points" and (ii) at least 30 one "tethering attachment point." A "backbone attachment point" as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct 115 WO 2004/094345 PCT/US2004/011822 from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, 5 e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring. Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA 10 molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid 15 binding proteins (e.g., during RISC formation and strand separation). SAccordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-l) is incorporated into at least one of said strands. R

R

6

R

2

XR

5

R

3 Z

R

4 20 (R-l) Referring to formula (R-1), X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CRRo, or absent; and Z is CRitRI 2 or absent. 25 Each of R', R 2 , R 3 , R 4 , R 9 , and R 1 0 is, independently, H, OR, ORb, (CH 2 )nORa, or

(CH

2 )nORb, provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R' 0 is ORa or ORb and that at least one of R 1 , R 2 , R 3,

R

4 ,

R

9, and R 10 is (CH 2 )nORa, or (CH 2 )nORb (when the RRMS is terminal, one of R', R 2 , R , R 4 , R 9 , and R l0 will include Ra and one will include Rb; when the RRMS is 116 WO 2004/094345 PCT/US2004/011822 internal, two of R', R 2 , R 3 , R 4 , R 9 , and R' 0 will each include an Rb); further provided that preferably ORa may only be present with (CH 2 )nORb and (CH 2 )nORa may only be present with ORb. Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, C 1

-C

6 alkyl optionally substituted with 5 1-3 R 13 , or C(O)NIHR 7 ; or R 5 and R" together are C 3

-C

8 cycloalkyl optionally substituted with

RI

4 . R7 is C 1

-C

20 alkyl substituted with NRcRd; R 8 is C 1 -C6 alkyl; R1 3 is hydroxy, C 1

-C

4 alkoxy, or halo; and R 14 is NRR 7 . 10 Ra is: A -P-B I C ; and Rb is: A I I -P-O-Strand I C 15 Each of A and C is, independently, O or S. B is OH, O, or 0 0 O-P-O-P-OH 0 PO I-OH O" O 20 R is H or C1-C6 alkyl; Rd is H or a ligand; and n is 1-4. In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is

N(CO)R

7 or NR 7 , Y is CR 9

R

10 , and Z is absent. 117 WO 2004/094345 PCT/US2004/011822 In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R7 orNR 7, Y is CRRo, and Z is CR"R 1 2 In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is 81 12

N(CO)R

7 or NR 7 , Y is NR 8 , and Z is CRuR 12 5 In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R 7 or NR 7 , Y is O, and Z is CR1"R1. In other preferred embodiments the ribose is replaced with a decalin scaffold, and X isCH 2 ; Y is CR 9 Ri 0 ; and Z is CR R 12; and R and R"1 together are C 6 cycloalkyl. In other preferred embodiments the ribose is replaced with a decalin/indane scaffold and, 10 and X is CH 2 ; Y is CRRio; and Z is CR1 'R12; and R 5 and R" 1 together are C 5 cycloalkyl. In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold. RRMSs described herein may be incorporated into any double-stranded RNA-likc molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the 15 sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the 20 antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and at (or within 1, 2, or 3 25 positions of) the 3' end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both ligands are located at the same end of the iRNA agent. In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the 30 hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual 118 WO 2004/094345 PCT/US2004/011822 tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a "hairpin ligand." In other embodiments, an iRNA agent may have an RRMS at the 3' end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an 5 RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand. In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, 10 have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA 15 agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length 20 being staggered. 5' ends are preferably phosphorylated. An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrier includes two "backbone attachment points" (hydroxyl groups), a "tethering attachment point," and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5' or 3' terminal subunit of the RNA 25 molecule, i.e., one of the two "W" groups may be a hydroxyl group, and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the RRMS may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA molecule, e.g., an iRNA agent. 30 The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG 4) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies. 119 WO 2004/094345 PCT/US2004/011822 The RRMS monomers generally include two differently functionalized hydroxyl groups

(OFG

1 and OFG 2 above), which are linked to the carrier molecule (see A in FIG 4), and a tethering attachment point. As used herein, the term "functionalized hydroxyl group" means that the hydroxyl proton has been replaced by another substituent. As shown in representative 5 structures B and C, one hydroxyl group (OFG 1 ) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG 2 ) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time 10 that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a "precursor" RRMS after a "precursor" RRMS monomer has been incorporated into the strand. 15 The (OFG 1 ) protecting group may be selected as desired, e.g., from T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, -OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl 20 groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A preferred protecting group is the dimethoxytrityl group. 25 When the OFG 2 in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG 1 is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or 30 oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG 2 . Subsequent to this operation, OFG 1 can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). R' can be substituted or unsubstituted alkyl or alkenyl. In 120 WO 2004/094345 PCT/US2004/011822 preferred embodiments, R' is methyl, allyl or 2-cyanoethyl. R" may a CI-Clo alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

OFG

2 in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be 5 any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the 10 monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be -C(O)(CH 2 )qC(O)-, or -C(O)(CH 2 )qS-, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can 15 not be used in conjunction with fluoride labile 5' silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride stable polystyrene based supports or PEG are preferred. Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R' or R 2 ; R3 or R 4 ; or R 9 and R I 0 if Y 20 is CR 9

R

1 o (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R4 and R 9 . Preferred tethering attachment points include R ; R 5 or R 6 when X is CH 2 .The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or 25 R2; R or R4; or R 9 or R 1 o (when Y is CR 9 RIo), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.) R

R

6 R2 X R R3- Z

R

4 121 WO 2004/094345 PCT/US2004/011822 (R-3) X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CRR'io; and Z is CR 1 1R 12 or absent. 5 Each of R', R 2 , R 3 , R 4 , R 9 , and R 1 0 is, independently, H, OR a , or (CH 2 )nORb, provided that at least two of R 1 , R 2 , R , R 4 , R 9 , and R 1 o are ORa and/or (CH 2 )nOR". Each of R s , R 6 , R" , and R 12 is, independently, a ligand, H, C1-C 6 alkyl optionally substituted with 1-3 R 13 , or C(0)NHR 7 ; or R 5 and R 1 together are C 3

-C

8 cycloalkyl optionally substituted with R 14 10 R 7 is H, a ligand, or C 1

-C

2 0 alkyl substituted with NRCRd; R 8 is H or CI-C 6 alkyl; R 13 is hydroxy, CI-C 4 alkoxy, or halo; R 14 is NRR7; R 15 is Ci-C 6 alkyl optionally substituted with cyano, or C 2

-C

6 alkenyl; R 1 6 is C 1 -Clo alkyl; and R 17 is a liquid or solid phase support reagent. L is -C(O)(CH2)qC(O)-, or -C(O)(CH 2 )qS-; Ra is CAr 3 ; Rb is P(O)(O)H, P(OR's)N(R16) 2 or L-R" 7 ; RC is H or CI-C 6 alkyl; and Rd is H or a ligand. 15 Each Ar is, independently, C 6

-C

0 lo aryl optionally substituted with C,-C 4 alkoxy; n is 1-4; and q is 0-4. Exemplary carriers include those in which, e.g., X is N(CO)R 7 or NR 7 , Y is CRR'o, and Z is absent; or X is N(CO)R 7 or NR 7 , Y is CR'R 10 , and Z is CR" R1 2 ; or X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR'"R12; or X is N(CO)R 7 or NR 7 , Y is O, and Z is CR"1R1 2 ; or X is CH 2 ; Y is 20 CR 9 Rio; Z is CR"R 1 2 , and Rs and R1 together form C 6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH 2 ; Y is CR 9 Ri 0 ; Z is CR"R 1 2 , and R 5 and R" together form C 5 cycloalkyl (H, z= 1). In certain embodiments, the carrier may be based on the pyrroline ring system or the 3 hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9

R

1 o, and Z is absent (D). OFG 1 25 is preferably attached to a primary carbon, e.g., an exocyclic alkylene 122 WO 2004/094345 PCT/US2004/011822

OFG

2

C

4 C3 CH 2

OFG

1 NC2 LIGAND D group, e.g., a methylene group, connected to one of the carbons in the five-membered ring ( CH2OFG 1 in D). OFG 2 is preferably attached directly to one of the carbons in the five membered ring (-OFG 2 in D). For the pyrroline-based carriers, -CH 2

OFG

1 may be attached to C 5 2 and OFG 2 may be attached to C-3; or -CH 2

OFG

1 may be attached to C-3 and OFG 2 may be attached to C-4. . In certain embodiments, CH 2

OFG

1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.For the 3-hydroxyproline-based carriers, -CH 2

OFG

1 may be attached to C-2 and OFG 2 may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is o10 restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH 2

OFG

1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric 15 forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen. 123 WO 2004/094345 PCT/US2004/011822 In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is

N(CO)R

7 or NR 7 , Y is CR 9

R

1 0 , and Z is CRUR 12 . OFG 1 is preferably

OFG

2 C4/ 'C3 -- (CH 2 )nOFG 1 NC2 LIGAND E 5 attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [-(CH 2 )nOFG' in E]. OFG 2 is preferably attached directly to one of the carbons in the six-membered ring (-OFG 2 in E). -(CH 2 )nOFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, o10 (CH 2 )nOFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH 2 )nOFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH 2 )nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-2;

-(CH

2 )nOFG 1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH 2 )nOFG may be, attached to C-4 and OFG 2 may be attached to C-3. The piperidine-based monomers may 15 therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH 2 )nOFG' and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, 20 single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen. 124 WO 2004/094345 PCT/US2004/011822 In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R or NR 7 , Y is NR , and Z is CR"R 12 , or the morpholine ring system (G), e.g., X is

N(CO)R

7 or NR 7 , Y is O, and Z is CR"R 12

.OFG

1 is preferably R"'1

OFG

2 OFG 2 N O C3 C3

CH

2 0FG' CH 2 0FG C2 C 2 LIGAND LIGAND F G 5 attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH 2 OFG' in F or G). OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG 2 in F or G). 10 For both F and G, -CH 2 OFG' may be attached to C-2 and OFG 2 may be attached to C-3; or vice versa. In certain embodiments, CH 2

OFG

1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH 2

OFG

1 and 15 OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R"' can be, e.g., CI-C 6 alkyl, preferably CH 3 . The 20 tethering attachment point is preferably nitrogen in both F and G. In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is

CH

2 ; Y is CR 9 R'io; Z is CR" R 12 , and R 5 and R 1 together form C 6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH 2 ; Y is CR 9 R'o; Z is CR"R 1 2 , and R 5 and Rn together form Cs cycloalkyl (H, z = 1). OFG' is preferably attached to a primary carbon, 125 WO 2004/094345 PCT/US2004/011822

OFG

2 ,07-0/05C C6 /C4 z( -- (CH2)nOFG 1 C2 H e.g., an exocyclic methylene group (n=l) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [-(CH 2 )nOFG' in H]. OFG 2 is preferably attached directly to one of C-2, C-3, C-4, 5 or C-5 (-OFG 2 in H). -(CH 2 )nOFG' and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups maybe attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, -(CH 2 )nOFG' and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CHI 2 )nOFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH 2 )nOFG 1 may be attached to C-3 and OFG 2 may be 10 attached to C-2; -(CH 2 )nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or (CH 2 )nOFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; -(CH 2 )nOFG' may be attached to C-4 and OFG 2 may be attached to C-5; or -(CH 2 )nOFG 1 may be attached to C-5 and

OFG

2 may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular 15 linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH 2 ),OFG' and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers 20 are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7. Other carriers may include those based on 3-hydroxyproline (J). Thus, -(CH 2 )nOFG' and

OFG

2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers 126 WO 2004/094345 PCT/US2004/011822 2

GFO(CH

2 ) _ OFG 1 N LIGAND J and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. 5 The tethering attachment point is preferably nitrogen. Representative carriers are shown in FIG. 5. In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any CI-Co 100 carbon-containing moiety, (e.g. C 1

-C

75 , C 1 10 C 50 , C 1

-C

20 , C 1

-C

10 , C 1

-C

6 ), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP

(CH

2 ) ; TAP-C(O)(CH2 f; or TAP-NR .... (CH2jNl , in which n is 1-6 and R.."".. is Ci

C

6 alkyl. and R d is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a 15 terminal oxyamino group, e.g., -ONH 2 , or hydrazino group, -NHNH 2 . The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP- CH),NH(LIGAND), TAP-C(O)(CH) NH(LIGAND), or TAP-NR .... (CH),nNH(LIGAND); 20 TAP-(CH)ONH(LIGAND), TAP-C(0)(CHz),ONH(LIGAND), or TAP-NR"" (CH ONH(LIGAND); TAP-(CH.),NHNH(LIGAND), TAP-C(O)(CHz)~NHNH(LIGAND), or TAP-NR .... ("CH-)nNHNH(LIGAND). In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl 25 halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NTIS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP 127 WO 2004/094345 PCT/US2004/011822 (CH C2) CIQ; TAP-C(O)(CH ?)CHO; or TAP-NR "(CH]CHO, in which n is 1-6 and R'"' is

CI-C

6 alkyl; or TAP-(CH)C()O1INHS; TAP-C(0)(CH C(O)ONHS; or TAP-NR"" .... (CHmC(O)ONHS, in which n is 1-6 and R"" is Ci-C 6 alkyl; TAP-(CH2 C(O)OCF 5 ; TAP-C(O)(CH),C(O) OC6F5; or TAP-NR .... (CH)1nC(O) OC 6 F-, in 5 which n is 1-6 and R.."".. is CI-C 6 alkyl; or -(CH nCH 2 LG; TAP-C(O)(CH L)nCIgLG; or TAP NR"" .... (CHCH 2 LG, in which n is 1-6 and R.."".. is C 1

-C

6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether. 10 Tethered Entities A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent. Preferred entities are those which target to the kidney, and also those that specifically target to tissues other than the kidney. 15 Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a "precursor" 20 RRMS after a "precursor" RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH 2 )nNH 2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can 25 subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether. In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a 30 cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid. Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a 128 WO 2004/094345 PCT/US2004/011822 polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; 5 and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may 10 also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2 hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl 15 alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide. 20 Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated 25 polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic 30 hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03 (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide 129 WO 2004/094345 PCT/US2004/011822 conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, 5 imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP. Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone 10 receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB. The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA 15 agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the iRNA agent into the cell by activating an 20 inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of 25 the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. 30 A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. 130 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed. 5 In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand. In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by 10 unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B 12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL). In another aspect, the ligand is a cell-permeation agent, preferably a helical cell 15 permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase. 20 The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be 25 about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 2, for example). 131 WO 2004/094345 PCT/US2004/011822 Table 2. Exemplary Cell Permeation Peptides Cell Amino acid Sequence Reference Permeation Peptide Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 1) Derossi et al., J. Biol. Chem. 269:10444, 1994 Tat fragment GRKKRRQRRRPPQC (SEQ ID NO:2) Vives et al., J. Biol. (48-60) Chem., 272:16010, 1997 Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Sequence- (SEQ ID NO:3) Biochem. Biophys. based peptide Res. Commun., 243:601, 1998 PVEC LLIILRRRIRKQAHAHSK (SEQ ID NO:4) Elmquist et al., Exp. Cell Res., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB (SEQ ID NO:5) J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA (SEQ ID NO:6) Oehlke et al., Mol. model peptide Ther., 2:339, 2000 Arg 9 RRRRRRRR (SEQ ID NO:7) Mitchell et al., J. Pept. Res., 56:318, 2000 Bacterial cell KFFKFFKFFK (SEQ ID NO:8) wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO:9) Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ IDNO:10) a-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO:11) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO:12) Bactenecin RKCRIVVIRVCR (SEQ ID NO:13) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP RFPPRFPGKR-NH2 (SEQ ID NO:14) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID NO:15) A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or 5 Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked 132 WO 2004/094345 PCT/US2004/011822 peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:16). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 17)) containing a 5 hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:18)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:19)) have been found to be capable of functioning as 10 delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide 15 moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD 20 peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA 25 agent to a tumor cell expressing cVB 3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an avl3 3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one 30 can use other moieties that target the ,av-3 3 integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein. A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell 133 WO 2004/094345 PCT/US2004/011822 permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., a -defensin, 0-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell 5 permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003). In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a helical peptide. Exemplary amphipathic a-helical peptides include, but are not limited to, 10 cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H 2 A peptides, Xenopus peptides, esculentinis 1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized 15 (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i d- 3, or i ±- 4 positions can provide stability. For example, cationic residues such as 20 lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate. Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; ox, 13, or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, 25 carbamate, or sulfonyl urea linkages; or cyclic peptides. Methods for making iRNA agents The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drug 30 Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001. In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the 134 WO 2004/094345 PCT/US2004/011822 c-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a "carbonyl donor," e.g., phenylchloroformate. 5 In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren 10 et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, FL, 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide. In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or 15is peptidomimetic tethered to an RRMS) can be a #-peptide. fl-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. 0-peptides are resistant to degradation by proteolytic enzymes. fl-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a /-peptide. 20 In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate. In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or 25 peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating 30 unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, 135 WO 2004/094345 PCT/US2004/011822 a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate). The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, 5 RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be 10 affiliated with a complex, such as a peptide-carrier complex. A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting 15 molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier. A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be 20 tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are 25 susceptible to enzymatic cleavage in the lysosomes. Targeting The iRNA agents of the invention are particularly useful when targeted to the kidney. An iRNA agent can be targeted to the kidney by incorporation of an RRMS containing a ligand that targets the kidney. 30 A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, can be useful. These agents target, for example, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also 136 WO 2004/094345 PCT/US2004/011822 be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting. Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to a non-kidney tissue, such as the liver. 5 An iRNA agent targeted to the kidney by an RRMS targeting moiety described herein can target a gene expressed in the kidney. Ligands on RRMSs can include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A. Palindromes 10 The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having a palindrome structure as described herein and those described in one or more of United States Provisional Application Serial No. 60/452,682, filed March 7, 2003; United States Provisional Application Serial No. 60/462,894, filed April 14,2003; and International Application No. PCT/US04/07070, filed March 8, 2004, all of which are hereby 15 incorporated by reference. The iRNA agents of the invention can target more than one RNA region. For example, an iRNA agent can include a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. The first and second sequences of the iRNA agent can be on different RNA strands, and 20 the mismatch between the first and second sequences can be less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent can be in bimolecular form. The first and second sequences of the iRNA agent can be fully complementary to each other. 25 The first target RNA region can be encoded by a first gene and the second target RNA region can encoded by a second gene, or the first and second target RNA regions can be different regions of an RNA from a single gene. The first and second sequences can differ by at least 1 nucleotide. The first and second target RNA regions can be on transcripts encoded by first and 30 second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example. The first target RNA region can include a nucleotide substitution, insertion, or deletion relative to the second target RNA region, or the second target RNA region can a mutant or variant of the first target region. 137 WO 2004/094345 PCT/US2004/011822 The first and second target RNA regions can comprise viral or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In addition, the first and second target RNA regions can correspond to hot-spots for genetic variation. 5 The compositions of the invention can include mixtures of iRNA agent molecules. For example, one iRNA agent can contain a first sequence and a second sequence sufficiently complementary to each other to hybridize, and in addition the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture can also include at least one additional iRNA agent variety that includes a 10 third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence can be sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. The first and second sequences can be on the same or different RNA strands, and the third 15 and fourth sequences can be on the same or different RNA strands. The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. The target RNA regions can correspond to genetic hot spots. 20 Methods of making an iRNA agent composition can include obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region can be obtained or provided, where each RNA target corresponds to a different variant or 25 mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent can be constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S), and the first and second sequences can be sufficiently complementary to hybridize. 30 Sequence analysis, e.g., to identify common mutants in the target gene, can be used to identify a region of the target gene that has high variability or mutational frequency. A region of the target gene having high variability or mutational frequency can be identified by obtaining or providing genotype information about the target gene from a population. 138 WO 2004/094345 PCT/US2004/011822 Expression of a target gene can be modulated, e.g., downregulated or silenced, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target 5 RNA region. An iRNA agent can include a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region. The first and second variant RNA target regions can correspond to first and second variants or mutants of a target gene, e.g., viral gene, tumor suppressor or oncogene. The first and second variant target 10 RNA regions can include allelic variants, mutations (e.g., point mutations), or polymorphisms of the target gene. The first and second variant RNA target regions can correspond to genetic hot spots. A plurality of iRNA agents (e.g., a panel or bank) can be provided. 15 Other than Canonical Watson-Crick Duplex Structures The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand, such as those described herein and those described in United States Provisional Application Serial No. 60/465,665, filed April 25, 20 2003, and International Application No. PCT/US04/07070, filed March 8, 2004, both of which are hereby incorporated by reference. The use of "other than canonical Watson-Crick pairing" between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in 25 the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the 30 position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense 139 WO 2004/094345 PCT/US2004/011822 targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA). In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex 5 between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound 10 by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence). Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence 15 from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are 20 sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can e, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex. Thus, in another aspect, the invention features, an iRNA agent which includes: a first 25 sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target 30 region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence. 140 WO 2004/094345 PCT/US2004/011822 Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA 5 duplex. The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex. A constrained or selected site can be present at a number of positions in the iRNA lo agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region. In some embodiment the duplex region of the iRNA agent will have, mismatches, in 15 addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent 20 sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. The monomers can be selected such that: first and second monomers are naturally occurring ribonuceotides, or modified ribonucleotides having naturally occurring bases, and when occupying complemetary sites either do not pair and have no substantial level of H 25 bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a 30 canonical Watson-Crick pairing with the base in the complemetary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atas, G-Aci, and GU. 141 WO 2004/094345 PCT/US2004/011822 The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed 5 by the monomer one (or both) of the sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they 10 otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one ( or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson Crick paring. The inclusion of such a monomers will have one or more of the following effects: it will 15 destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized. By way of example: The monomer at the selected site in the first sequence includes an A (or a modified base 20 which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical 25 Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has 30 a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is 142 WO 2004/094345 PCT/US2004/011822 chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which 5 will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be 10 useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. Anon-naturally occurring or modified monomer or monomers can be chosen such that 15 when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they 20 either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA 25 agent. An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and 30 the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 6. The pair shown in FIG. 6 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing 143 WO 2004/094345 PCT/US2004/011822 moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by 5 reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C. iRNA agents of the invention can include: A sense sequence, which preferably does not target a sequence in a subject, and an anti 10 sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that: 15 The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer 20 in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner); The monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target 25 stand; Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence. The inclusion of such a monomers will have one or more of the following effects: it will 30 destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized. 144 WO 2004/094345 PCT/US2004/011822 The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex. A constrained or selected site can be present at a number of positions in the iRNA agent 5 duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region. In some embodiment the duplex region of the iRNA agent will have, mismatches, in 10 addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent 15 sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. The monomers can be selected such that: first and second monomers are naturally occurring ribonuceotides, or modified ribonucleotides having naturally occurring bases, and when occupying complemetary sites either do not pair and have no substantial level of H 20 bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a 25 canonical Watson-Crick pairing with the base in the complemetary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Ais, and GU. 30 The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences With the respective target sequence. The 145 WO 2004/094345 PCT/US2004/011822 monomer is one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences-are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they 5 otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson Crick paring. The inclusion of such a monomers will have one or more of the following effects: it will 10 destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex' interactions between the a sequence and the intended target will not be destabilized. By way of example: The monomer at the selected site in the first sequence includes an A (or a modified base 15 which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical 20 Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has 25 a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is 30 chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which 146 WO 2004/094345 PCT/US2004/011822 will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is 5 chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand. 10 Anon-naturally occurring or modified monomer or monomers can be chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second 15 monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with 20 a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent. An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. When placed in complementary positions of the iRNA agent these monomers will pair 25 very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. The monomer at the selected position in the sense strand can be a universal pairing 30 moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases 147 WO 2004/094345 PCT/US2004/011822 the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C. iRNA agents of the invention can include: A sense sequence, which preferably does not target a sequence in a subject, and an anti 5 sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that: 10 The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer 15 in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner); The monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target 20 stand; Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence. The inclusion of such a monomers will have one or more of the following effects: it will 25 destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized. The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, 30 but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex. A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in 148 WO 2004/094345 PCT/US2004/011822 the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region. The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein. 5 In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein. E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. o10 In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length. In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. 15 Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. 20 One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a 25 non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer 30 forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a 149 WO 2004/094345 PCT/US2004/011822 canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm. By way of example: 5 the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G; the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is 10 chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G; the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, 15 Atrans, or U; or the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing. In another embodiment a non-naturally occurring or modified monomer or monomers is 20 chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H 25 bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent. 30 An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2 150 WO 2004/094345 PCT/US2004/011822 thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and 5 preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C. 10 In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., 15 under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differe in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the, 20 anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein. In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or 25 a viral genome, e.g., the HCV genome. An embodiment is illustrated in the FIGs. 7 and 8. In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained positoin in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in 30 a duplex between a sequence form the iRNA agent and another RNA, e.g., a taret sequence. The determinations can be compared. An iRNA agent thus analysed can be used in the devolopement of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or desing optimized iRNA agents. 151 WO 2004/094345 PCT/US2004/011822 In another aspect, the invention features, a kit which inlcudes one or more of the folowing an iRNA described herein, a sterile container in which the iRNA agent is discolsed, and instructions for use. In another aspect, the invention features, an iRNA agent containing a constrained 5 sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods 10 described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein. 15 The term "other than canonical Watson-Crick pairing" as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to 20 the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or(3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less 25 strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C. The term "off-target" as used herein, refers to as a sequence other than the sequence to be silenced. Universal Bases: "wild-cards"; shape-based complementarity 30 Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by 'inverse' sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926) 152 WO 2004/094345 PCT/US2004/011822 F CH 3

H

3 C H N HO /..F HO N OF 00 OH F OH (Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632) 5 (Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192) F

H

3 C HO F 0 OH (Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057) 10 (Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656) F

CH

3

H

3 C N HO H F HO N 0 OH F OH z 15 153 WO 2004/094345 PCT/US2004/011822 NO2 HO OH 3-nitropyrrole /N NO 2 HON OH 5-nitroindole N O N 0 N 0 N 0 MICS PIM SMICS ( (Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; 5 McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914) O O 0 0 TM DM ° ICS PICS N 2MN DMN 7AI 2Np 3MN (1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural 10 hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804) 154 WO 2004/094345 PCT/US2004/011822 N N 7AI (Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J Am. Chem. Soc., 2001, 123, 7439-7440) N HO. 0 OH 5 (1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586) 10 (A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014) 15 (2,2'-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)

NH

2 NH 2 NN N HO HO 0 OH OH d2APy d2APm (Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. . Am. 20 Chem. Soc., 2000, 122, 6512-13) 155 WO 2004/094345 PCT/US2004/011822

NO

2 HO N OH (Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2'-deoxy-13-D-ribofuranosyl)-3 nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal 5 nucleosides: 1-(2'-deoxy-p3-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209) OH H OH SN-H ........... o O A\ I ... .,,N / HO N' B u HO N "H- HON 0 "JN HH H O OH Bu H N-O O0 N N ,, (Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a 10 CG base-pair in an organic solvent. Zimnmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770) /-0 0 o DNA 0 ONO 0 N02 01 DNA (A universal, photocleavable DNA base: nitropiperonyl 2'-deoxyriboside. . Org. Chem., 2001, 66, 2067 2071) 156 WO 2004/094345 PCT/US2004/011822 o R-- N H 0 HN I N N H (Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine 5 bulge as evidenced by photooxidation of GC doublet, Nakatani, K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chemin. Lett., 2001, 11, 335-337) 0 N- H,,,,O N-/ \ ~ 0, N H-N N -0/ 0 0 0 00 0 Asymmetrical Modifications The monomers and methods described herein can be used to prepare an RNA, e.g., an 10 iRNA agent, can be asymmetrically modified as described herein, and as described in International Application Serial No. PCT/USO4/07070, filed March 8, 2004, which is hereby incorporated by reference. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetricalmodification is a modification found 15 on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; 20 modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification 157 WO 2004/094345 PCT/US2004/011822 as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting. While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the 5 different or "asymmetrical" functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand. In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense 10 strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2' modification, e.g., 2' OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5' or 3' end of the sense strand of the iRNA agent. In 15 a preferred example, a P of the backbone is replaced with S in the antisense strand, 2'OMe is present in the sense strand, and a targeting moiety is added to either the 5' or 3' end of the sense strand of the iRNA agent. In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense 20 strand has a modification which is not found on the sense strand. Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification 25 which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence. In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. 30 E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand. In an embodiment one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an 158 WO 2004/094345 PCT/US2004/011822 asymmetrical 2' OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2'-O alkyl, preferably, 2'-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the 5 antisense strand. There can be one or multiple 2'-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2' OMe modifications on the sense strand and multiple phophorothioate modifications on the 10 antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3' overhangs of about 2 subunits in length. Asymmetrical modifications are useful for promoting resistance to degradation by 15 nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are 20 relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety 25 on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2', 6', 7', 30 or 8' position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as 159 WO 2004/094345 PCT/US2004/011822 those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3' overhang, e.g., at the 3' terminus, and on the 5 antisense strand, e.g., in a 3' overhang, e.g., at the 3' terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand. Examples of modifications which inhibit exonucleolytic degradation can be found herein. 10 Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3' overhang, e.g., at the 3' 15 terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3' overhang, e.g., at the 3' terminus; modification with naproxene, ibuprofen, or other moieties which inhibit 20 degradation at the 3' terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by 25 inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive 30 charge or one negative charge) can be placed on the sense strand. Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids ,2 160 WO 2004/094345 PCT/US2004/011822 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand. iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3' 5 ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5' end of the sense strand and the 3'end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following 10 are used: modifications of the 5' end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5' exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a "tight" structure at this end of the molecule. 15 The end region of the iRNA agent defined by the 3' end of the sense strand and the 5'end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an "open" structure at this end of the 20 molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments. Exemplary modifications for asymmetrical placement in the sense strand include the following: 25 (a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S); these modifications can be used to promote nuclease resistance; (b) 2'-O alkyl, e.g., 2'-OMe, 3'-O alkyl, e.g., 3'-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding 30 of the sense to the antisense strand, the 3' modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC; (c) 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe and with P=O or P=S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the 161 WO 2004/094345 PCT/US2004/011822 antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC; (d) L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the 5 antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC; (e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of 10 the sense strand to avoid sense strand activation by RISC; (f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand; (g) cationic groups and Zwitterionic groups (preferably at a terminus), these 15 modifications can be used to promote nuclease resistance; (h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC. 20 Exemplary modifications for asymmetrical placement in the antisense strand include the following: (a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S); (b) 2'-O alkyl, e.g., 2'-OMe, (at terminal positions); 25 (c) 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe) e.g., terminal at the 3' end); e.g., with P=O or P=S preferably at the 3'-end, these modifications are preferably excluded from the 5' end region as they may interfere with RISC enzyme activity such as kinase activity; (d) L sugars (e.g, L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); e.g., terminal at the 3' end; e.g., with P=O or P=S preferably at the 3'-end, these modifications are preferably 30 excluded from the 5' end region as they may interfere with kinase activity; (e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5' end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a "loose" structure in the 5' region, additionally, they may interfere with kinase activity; 162 WO 2004/094345 PCT/US2004/011822 (f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines); (g) cationic groups and Zwitterionic groups (preferably at a terminus); conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, 5 ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred. The 5'-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3' end, particularly in the 3' overhang. 10 In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity. The decrease in activity can be less than a preselected level of decrease. In 15 preferred embodiments decrease in activity means a decrease of less than'5, 10, 20, 40, or 50 % activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity. The optimized property can be any property described herein and in particular the 20 properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2'-O alkyl modifications, e.g., 2'-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate 25 modifications, in the antisense sequence. In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications. 30 Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified. Table 3 shows examples having strand I with a selected modification and strand II with a selected modification. 163 WO 2004/094345 PCT/US2004/011822 Table 3. Exemplary strand I- and strand II-modifications Strand I Strand II Nuclease Resistance (e.g., 2'-OMe) Biodistribution (e.g., P=S) Biodistribution conjugate Protein Binding Functionality (e.g., Lipophile) (e.g., Naproxen) Tissue Distribution Functionality Cell Targeting Functionality (e.g., Carbohydrates) (e.g., Folate for cancer cells) Tissue Distribution Functionality Fusogenic Functionality (e.g., Kidney Cell Targetingmoieties) (e.g., Polyethylene imines) Cancer Cell Targeting Fusogenic Functionality (e.g., RGD peptides and imines) (e.g., peptides) Nuclease Resistance (e.g., 2'-OMe) Increase in binding Affinity (5-Me-C, 5-Me-U, 2 Nuclease Resistance (e.g., 2'-OMe) ti-,2aioA -lmLA thio-U, 2-amino-A, G-clamp, LNA) Tissue Distribution Functionality RISC activity improving Functionality Helical conformation changing Tissue Distribution Functionality Functionalities (P=S; lipophile, carbohydrates) Z-X-Y Architecture 5 The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having a Z-X-Y architecture or structure such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/510,246, filed on October 9, 2003, which is hereby incorporated by reference, copending, co-owned United States Provisional Application Serial No. 60/510,318, filed on October 10, 2003, which is 10 hereby incorporated by reference, and copending, co-owned International Application No. PCT/US04/07070, filed March 8, 2004. Thus, an iRNA agent can have a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region: 15 Z-X-Y. 164 WO 2004/094345 PCT/US2004/011822 It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X. 5 The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra 10 strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment ofa Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3' or 5' terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical 15 inter-subunit bonds in the Z region include: P=O; P=S; S-P=S; P-NR 2 ; and P-BR 2 . Chiral P=X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3'-OR, 3'SR, 2'-OMe, 3'-OMe, and 2'OH modifications and moieties; alpha configuration bases; and 2' arabino modifications. 20 The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though 25 other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2'-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments 30 have 4 or more consecutive 2'OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2'OH subunits. The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of 165 WO 2004/094345 PCT/US2004/011822 modifications can be present, and infact, one will usually be a 3' overhang and one will usually be a 5' overhang. In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe or other alkyl; and an X 5 region that includes at least four consecutive ribonucleoside subunits in which the 2'-OH remains unsubstituted. The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications 10 can be provided bewteen the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided. In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe; and an X region that 15 includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2'-OH remains unsubstituted. As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in 20 particular it is preferred that consecutive modified linkages be avoided. Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is 25 joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA. It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although 30 the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2'-hydroxyl 166 WO 2004/094345 PCT/US2004/011822 ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA. In a preferred embodiment Y and /or Z will be free of phosphorothioate linkages, though 5 either or both may contain other modifications, e.g., other modifications of the subunit linkages. In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2' moieties. In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages. 10 In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-O-alkyl moiety. E.g., any adjacent 2'-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage. 15 In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a 20 phosphorothioate linkage. In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides, are be connected by a linkage 25 other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage. In a prefered embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such 30 modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-O-alkyl moiety. E.g., any adjacent 2'-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2' moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages. 167 WO 2004/094345 PCT/US2004/011822 A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to 5 another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Yand /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other. 10 An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b 15 present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 20 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target. 25 Differential Modification of Terminal Duplex Stability In one aspect, the monomers and methods described herein can be used to prepare an iRNA agent having differential modification of terminal duplex stability (DMTDS). In addition, the monomers and methods described herein can be used to prepare iRNA agents having DMTDS and another element described herein. E.g., the monomers and methods 30 described herein can be used to prepare an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the kidney, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent 168 WO 2004/094345 PCT/US2004/011822 administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS. iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5' end of the 5 antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5'end. While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, 10 for example, promoting the effect of the enzyme in the proximity of the 5' end of the antisense strand. The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3' end of the antisense strand duplex. This can be 15 accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3' end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5'end. Modifications which increase the tendency of the 5' end of the duplex to dissociate can 20 be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3' end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5' end of the duplex to dissociate. 25 Decreasing the stability of the AS 5' end of the duplex Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: 30 A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine); 169 WO 2004/094345 PCT/US2004/011822 mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; pairings which include a universal base are preferred over canonical pairings. 5 A typical ds iRNA agent can be diagrammed as follows: S 5' R 1

N

1

N

2

N

3

N

4

N

5 [N] N- 5

N.

4

N.

3 N-2 N- 1

R

2 3' AS 3' R 3 Ni N 2

N

3

N

4

N

5 10 [N] N-5 N- 4

N-

3

N-

2

N-

1

R

4 5' S:AS P 1

P

2

P

3

P

4 Ps [N] P-5 P-4 P- 3

P-

2 P-1 5' 15 S indicates the sense strand; AS indicates antisense strand; R, indicates an optional (and nonpreferred) 5' sense strand overhang; R 2 indicates an optional (though preferred) 3' sense overhang; R 3 indicates an optional (though preferred) 3' antisense sense overhang; R 4 indicates an optional (and nonpreferred) 5' antisense overhang; N indicates subunits; [N] indicates that 20 additional subunit pairs may be present; and P,, indicates a paring of sense Nx and antisense Nx. Overhangs are not shown in the P diagram. In some embodiments a 3' AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3' S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.) 25 It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5' end of the AS strand. The terminal pair (the most 5' pair in terms of the AS strand) is designated as P.- 1 , and the subsequent pairing positions (going in the 3' direction in terms of the AS strand) in the duplex are designated, P.- 2 , P.- 3 , P-4, P- 5 , and so on. The preferred region in which to modify to modulate duplex formation is at P.-5 30 through P- 1 , more preferably P- 4 through P-.

1 , more preferably P- 3 through P-1. Modification at P 1, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of: 170 WO 2004/094345 PCT/US2004/011822 A:U G:U I:C mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings 5 which include a universal base. In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand. In a preferred embodiment the at least 2, or 3, of the pairs in P-.

1 , through P-4, are pairs 10 which promote disociation. In a preferred embodiment the at least 2, or 3, of the pairs in P- 1 , through P- 4 , are A:U. In a preferred embodiment the at least 2, or 3, of the pairs in P- 1 , through P- 4 , are G:U. In a preferred embodiment the at least 2, or 3, of the pairs in P.-, through P.- 4 , are I:C. In a preferred embodiment the at least 2, or 3, of the pairs in P- 1 , through P- 4 , are 15 mismatched pairs, e.g., non-canonical or other than canonical pairings pairings. In a preferred embodiment the at least 2, or 3, of the pairs in P- 1 , through P 4 , are pairings which include a universal base. Increasing the stability of the AS 3' end of the duplex Subunit pairs can be ranked on the basis of their propensity to promote stability and 20 inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability: 25 G:C is preferred over A:U Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C) 30 2-amino-A:U is preferred over A:U 2-thio U or 5 Me-thio-U:A are preferred over U:A G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G guanadinium-G-clamp:G is preferred over C:G 171 WO 2004/094345 PCT/US2004/011822 psuedo uridine:A is preferred over U:A sugar modifications, e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the 5 propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3' end of the AS strand. The terminal pair (the most 3' pair in terms of the AS strand) is designated as PI, and the subsequent pairing positions (going in the 5' direction in terms of the AS strand) in the duplex are designated, P 2 , P 3 , P 4 , P5, and so on. The preferred region in which to modify to modulate duplex formation is at P 5 through P 1 , more preferably P 4 through P 1 , more preferably 10 P 3 through Pi. Modification at P1, is particularly preferred, alone or with mdification(s) at other position(s), e.g.,any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of: 15 G:C a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C) 2-amino-A:U 2-thio U or 5 Me-thio-U:A 20 G-clamp (an analog of C having 4 hydrogen bonds):G guanadinium-G-clamp:G psuedo uridine:A a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding. 25 In a preferred embodiment the at least 2, or 3, of the pairs in P- 1 , through P- 4 , are pairs which promote duplex stability. In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P 4 , are G:C. In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P 4 , are a pair 30 having an analog that increases stability over Watson-Crick matches. In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are 2-amino A:U. In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are 2-thio U or 5 Me-thio-U:A. 172 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are G clamp:G. In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are guanidinium-G-clamp:G. 5 In a preferred embodiment the at least 2, or 3, of the pairs in PI, through P 4 , are psuedo uridine:A. In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhances binding. 10 G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, "The Synthesis of 2'-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction," Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., "Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-O-methyl 15 G-clamp ribonucleoside analogues," Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., "Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp," Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., "High Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al., "Synthesis of Amino- and Guanidino-G-Clamp PNA 20 Monomers," Organic Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2 aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties," Biochemistry, 41:1323-7, 2002; Flanagan, et al., "A cytosine analog that confers enhanced potency to antisense oligonucleotides," Proceedings Of The National Academy Of Sciences Of 25 The United States Of America, 96:3513-8, 1999. Simultaneously decreasing the stability of the AS 5'end of the duplex and increasing the stability of the AS 3' end of the duplex As is discussed above, an iRNA agent can be modified to both decrease the stability of 30 the AS 5'end of the duplex and increase the stability of the AS 3' end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5' end of the duplex with one or more of the stability increasing modifications in the AS 3' end of the duplex. Accordingly a preferred embodiment includes modification in P- through P- 1 , more preferably P- 4 through P.-1 and more preferably P.- 3 through P.-. Modification at P.-, is particularly 173 WO 2004/094345 PCT/US2004/011822 preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5' end of the duplex region be chosen independently from the group of: 5 A:U G:U I:C mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and 10 a modification in P 5 through P 1 , more preferably P 4 through P 1 and more preferably P 3 through P 1 . Modification at PI, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3' end of the duplex region be chosen independently 15 from the group of: G:C a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C) 20 2-amino-A:U 2-thio U or 5 Me-thio-U:A G-clamp (an analog of C having 4 hydrogen bonds):G guanadinium-G-clamp:G psuedo uridine:A 25 a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding. The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA 30 agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the AS strand, to provide a desired level of DMTDS. 174 WO 2004/094345 PCT/US2004/011822 The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P.

5 through P-1 and/or at least one P in P 5 through P 1 to provide a DMTDS iRNA agent. DMTDS iRNA agents can be used in any method described herein, e.g., to silence any 5 gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3' AS overhangs 10 and/or 3' S strand overhangs) which self associate to form intrastrand duplex structure. Preferably these iRNA agents will have an architecture described herein. Other Embodiments An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted 15 into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is 20 imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into sRNA agent fragments that mediate gene silencing. 25 In vivo Delivery An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery 30 vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, 175 WO 2004/094345 PCT/US2004/011822 that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to 5 adamantine which in turn is non-covalently associated with a cyclodextrin. The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems, as described in U.S. Patent No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear o10 cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand. A composition for iRNA delivery can employ an "inclusion complex," a molecular compound having the characteristic structure of an adduct. In this structure, the "host molecule" 15 spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the "guest molecule") is situated in the cavity of the host molecule without affecting the framework structure of the host. A "host" is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference. Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting 20 inclusion compounds belong to the class of "host-guest" complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing 25 agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent 30 having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell 176 WO 2004/094345 PCT/US2004/011822 contact (e.g., targeting or contact to a kidney cell), intercellular trafficking, and/or cell entry and release. Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, 5 from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified 10 with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety. Physiological Effects The iRNA agents described herein can be designed such that determining therapeutic 15 toxicity is made easier by the complementarity of the iRNA agent with both a human and a non human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, 20 monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can 25 be complementary to a human and more than one, e.g., two or three or more, non-human animals. The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect. 30 Delivery Module The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent described herein, that can be used with a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional 177 WO 2004/094345 PCT/US2004/011822 Application Serial No. 60/454,265, filed on March 12, 2003, and International Application Serial No. PCT/USO4/07070, filed March 8, 2004, both of which are hereby incorporated by reference. The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more 5 preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a 10 kidney cell. An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in 15 need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the kidney. The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent. 20 The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction), (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a 25 cell membrane, e.g., an endosome membrane), and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl 30 gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic. 178 WO 2004/094345 PCT/US2004/011822 Carrier agents The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would 5 preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.. The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier 10 molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2 hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl 15 alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods. A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at 20 a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent. Fusogenic agents A fusogenic agent of a modular complex described herein can be an agent that is 25 responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can 30 serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell. In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. 179 WO 2004/094345 PCT/US2004/011822 The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane. 5 The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI. In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. o10 In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin. A fusogenic agent can have one or more of the following characteristics: (a) is at least 1Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of 15 at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent. Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can 20 be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the 25 test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid 30 instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH 180 WO 2004/094345 PCT/US2004/011822 environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment. Condensing agent 5 The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing 10 agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide. A condensing agent can have the following characteristics: (a) at least 1Da in size; (b) 15 has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent. Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by 20 evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such 25 techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., 30 condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid. 181 WO 2004/094345 PCT/US2004/011822 Amphipathic Delivery Agents The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent described herein, that can be used with an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned United 5 States Provisional Application Serial No. 60/455,050, filed on March 13, 2003, and International Application Serial No. PCT/USO4/07070, filed March 8, 2004, which is hereby incorporated by reference. An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. o10 As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure. One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a 15 polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) "Protein design and folding: template trapping of self-assembled helical bundles" J Pept Sci 7(3):146-51; Chen et al. (2002) "Determination of stereochemistry stability 20 coefficients of amino acid side-chains in an amphipathic alpha-helix" J Pept Res 59(1):18-33; Iwata et al. (1994) "Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation" J Biol Chem 269(7):4928-33; Cornut et al. (1994) "The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin" 25 FEBS Lett 349(1):29-33; Negrete et al. (1998) "Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices," Protein Sci 7(6):1368-79. Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety 30 having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure. 182 WO 2004/094345 PCT/US2004/011822 The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, 5 penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, 10 o wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell. An amphipathic polymer useful in the compositions described herein has at least 2, 15 preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits. 20 The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase 25 the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell. In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent 30 or noncovalent linkages. The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment. The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and 183 WO 2004/094345 PCT/US2004/011822 one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a 5 hydrophobic face. The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an c-helical conformation as its secondary structure. In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or 10 one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2'-bis(substituted)- 1-1 '-binaphthyl or a 2,2'-bis(substituted) biphenyl. 15 A subunit may include an aromatic moiety of Formula (M): 184 WO 2004/094345 PCT/US2004/011822

R

3

R

4 R2 R4 R, R3 I I (M) 5 The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase 10 the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell. In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent 15 or noncovalent linkages. The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment. The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and 20 one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be 185 WO 2004/094345 PCT/US2004/011822 such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face. The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an oc-helical conformation as its secondary structure. 5 In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit 10 may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2'-bis(substituted)-l-1 '-binaphthyl or a 2,2'-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

R

3 R4 R2 R4 R,

R

3 15 (M) 186 WO 2004/094345 PCT/US2004/011822 Referring to Fonnula M, R 1 is C 1 -Co 10 o alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C 1

-C

0 lo 0 perfluoroalkyl; or OR 5 . 5 R 2 is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR 6 ; or C 1 -Cloo 00 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O),

SO

2 , alkenyl, or alkynyl. 10 R 3 is hydrogen, or when taken together with R 4 froms a fused phenyl ring.

R

4 is hydrogen, or when taken together with R 3 froms a fused phenyl ring.

R

5 is C 1 -Co 1 00 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or CI-Cloo 00 perfluoroalkyl; and R 6 is C 1 C 100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl 15 sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(0), SO2, alkenyl, or alkynyl. Increasing cellular uptake of dsRNAs 20 A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-KB. The drug can have a transient effect on 25 the cell. The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or 30 myoservin. The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon. 187 WO 2004/094345 PCT/US2004/011822 iRNA conjugates An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is 5 directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon. 10 iRNA PRODUCTION An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage. Organic Synthesis 15is An iRNA can be made by separately synthesizing each respective strand of a double stranded RNA molecule. The component strands can then be annealed. A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotI reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a 20 phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection. Organic synthesis can be used to produce a discrete iRNA species. The complementary 25 of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini. 188 WO 2004/094345 PCT/US2004/011822 dsRNA Cleavage iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used: 5 In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe T M RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates 10 are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNApolymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes. In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or 15 comparable RNAse III-based activity For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex ). See, e.g., Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9. and Hammond Science 2001 Aug 10;293(5532):1146-50. dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 20 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present. Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For 25 example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process. Synthesis of modified and nucleotide surrogate iRNA agents is discussed below. FORMULATION 30 The iRNA agents described herein can be formulated for administration to a subject For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, 189 WO 2004/094345 PCT/US2004/011822 that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less 5 than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA o10 composition is formulated in a manner that is compatible with the intended method of administration (see, below). In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and 15 other self-assembly. A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg 2 +), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as 20 RNAsin) and so forth. In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different 25 genes. In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the 30 treatment of a cancer might further comprise a chemotherapeutic agent. Exemplary formulations are discussed below: 190 WO 2004/094345 PCT/US2004/011822 Liposomes For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, 5 e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the 10 term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA 15 composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the 20 cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types, e.g., to cells of the kidney, such as those described herein. A liposome containing a iRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that 25 micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the 30 iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA. If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a 191 WO 2004/094345 PCT/US2004/011822 polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation. Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the 5 delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochimn. Biophys. Acta 775:169, 1984; Kim, et 10 al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 15 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes. Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid 20 molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274). One major type of liposomal composition includes phospholipids other than naturally 25 derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such 30 as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, 192 WO 2004/094345 PCT/US2004/011822 Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J 11:417,1992. In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able 5 to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages. Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from o10 metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact 15 spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA). A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be 20 used in combination with a phospholipid to form DNA-complexing vesicles. LipofectinTM Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the 25 resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the 30 oleoyl moieties are linked by ester, rather than ether linkages. Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TransfectamTM, Promega, Madison, Wisconsin) and 193 WO 2004/094345 PCT/US2004/011822 dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678). Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE (See, Gao, 5 X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other 10 commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194. Liposomal formulations are particularly suited for topical administration, liposomes 15 present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes 20 can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal ofDrug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 25 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/ 30 cholesterol/polyoxyethylene- 1 0-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder. Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can 194 WO 2004/094345 PCT/US2004/011822 be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less 5 than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome. 10 Surfactants For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Surfactants find wide 15 application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant. In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, 20 both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms," Marcel Dekker, Inc., New York, NY, 1988, p. 285). If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. 25 Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, 30 propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. 195 WO 2004/094345 PCT/US2004/011822 If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, 5 acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps. If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of 10 this class. If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. The use of surfactants in drug products, formulations and in emulsions has been reviewed 15 (Rieger, in "Pharmaceutical Dosage Forms," Marcel Dekker, Inc., New York, NY, 1988, p. 285). Micelles and other Membranous Formulations For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can 20 be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. "Micelles" are defined herein 25 as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic. A mixed micellar formulation suitable for delivery through transdermal membranes may 30 be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal Cs to C 22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, 196 WO 2004/094345 PCT/US2004/011822 monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The 5 micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles. In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then 10 mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing. Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the 15 formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition. For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under 20 pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray. 25 The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane). The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to 30 increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation. 197 WO 2004/094345 PCT/US2004/011822 Particles For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with 5 other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles 10 can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description. Sustained-Release Formulations. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA 15 agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time. 20 Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 pm and preferably larger than 20 am). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of 25 the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 fm rather than 10 /m. The time of mixing also can be altered. Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal 30 injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation. 198 WO 2004/094345 PCT/US2004/011822 Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres. 5 Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions 10 can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a 1/8" microtip probe, at setting #3. 15 Spray-Drying. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by 20 spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA. 25 Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996. Spray drying can include atomizing a solution, emulsion, or suspension to form a fine 30 mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected. 199 WO 2004/094345 PCT/US2004/011822 Spray drying includes bringing together a highly dispersed liquid, and a sufficient vohune of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a 5 current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size. Spray-dried powdered particles can be approximately spherical in shape, nearly uniform 10 in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being 15 aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common 20 and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chlorofonrm, freons, and hydrocarbons. Nitrogen gas and carbon dioxide 25 are also contemplated as a suitable blowing agent. Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that 30 generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques. 200 WO 2004/094345 PCT/US2004/011822 The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase 5 bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By "associated with or comprise" it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant. 10 Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and 15 selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations. 20 Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 400 C. Preferably, the incorporated lipids are relatively long chain (i.e. C 6

-C

22 ) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations 25 comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, 30 polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein. 201 WO 2004/094345 PCT/US2004/011822 Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (SpansTM 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and 5 sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic.RTM. F68), poloxamer 407 (Pluronic.RTM. F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In 10 preferred embodiments, the microstructures may comprise oleic acid or its alkali salt. In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA 15 agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3 dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3 (trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4'-trimethylammonio)butanoyl-sn glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated. For the spraying process, such spraying methods as rotary atomization, pressure 20 atomization and two-fluid atomization can be used. Examples of the devices used in these processes include "Parubisu [phonetic rendering] Mini-Spray GA-32" and "Parubisu Spray Drier DL-41", manufactured by Yamato Chemical Co., or "Spray Drier CL-8," "Spray Drier L-8," "Spray Drier FL-12 ," "Spray Drier FL-16" or "Spray Drier FL-20," manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer. 25 While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50oC to about 200oC, preferably between about 50oC and 100 0 C. The temperature of the outlet gas used to dry the sprayed 30 material, may vary between about 0OC and about 150oC, preferably between 0OC and 90oC, and even more preferably between 0OC and 60'C. The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the 202 WO 2004/094345 PCT/US2004/011822 resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 Am or less with about 90% of the mass being in particles having a diameter less than 5 pm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 [m with about 80% of the mass of the particles having a diameter of less than 5 pm. 5 The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the 10 dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling. Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be 15 formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in 20 the range from 25 /m to 100 /m. A preferred carrier material is crystalline lactose having a size in the above-stated range. Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or 25 other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers. Methods for spray drying hydrophobic and other drugs and components are described in 30 U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Phannrm. Acta Helv 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP 203 WO 2004/094345 PCT/US2004/011822 806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP 4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870. 5 LYOPHILIZATION. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which 10 water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the 15 incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and 20 size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention. Targeting For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that 25 these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or 30 precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the 204 WO 2004/094345 PCT/US2004/011822 surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer specific antigen of the kidney (e.g., G250, CA1 5-3, CA19-9, CEA, or HER2/neu) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting 5 moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof). Table 4 provides a number of antigens which can be used to target an iRNA to a selected cell, such as when targeting of the iRNA agent to a tissue other than the kidney is desired. 10 Table 4. Targeting Antigens ANTIGEN Exemplary tumor tissue CEA (carcinoembryonic antigen) colon, breast, lung PSA (prostate specific antigen) prostate cancer CA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancer HER2/neu breast cancer a-feto protein testicular cancer, hepatic cancer P3-HCG (human chorionic gonadotropin) testicular cancer, choriocarcinoma MUC-1 breast cancer Estrogen receptor breast cancer, uterine cancer Progesterone receptor breast cancer, uterine cancer EGFr (epidermal growth factor receptor) bladder cancer In one embodiment, the targeting moiety is attached to a liposome. For example, US Patent 6,245,427 describes a method for targeting a liposome using a protein or peptide. In 15 another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide. GENES AND DISEASES 20 In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes: providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by 25 cleavage, a gene which promotes unwanted cell proliferation; 205 WO 2004/094345 PCT/US2004/011822 administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject, thereby treating the subject. In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a 5 kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor. In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers. 10 In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer. In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., 15 colon cancers. In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers. In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used 20 to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma. In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia. 25 In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia. In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JNK expression, 30 e.g., pancreatic or breast cancers. In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia. 206 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the iRNA agent silences the Erkl/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erkl/2 expression, e.g., lung cancer. In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus 5 can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer. In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia. 10 In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma. In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, 15 e.g., ovarian, prostate or breast cancers. In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers. In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be 20 used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma. In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers. 25 In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers. In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, 30 e.g., breast cancer. In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers. 207 WO 2004/094345 PCT/US2004/011822 In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers. In another preferred embodiment the iRNA agent silences the WNT- 1 gene, and thus can 5 be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma. In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma. 10 In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma. In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC 15 expression, e.g., breast cancer. In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer. In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be 20 used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer. In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers. 25 In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer. In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted 30 topoisomerase I expression, e.g., ovarian and colon cancers. In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers. 208 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma. In a preferred embodiment the iRNA agent silences mutations in the p21 (WAF1/CIP 1) 5 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21 (WAFl /CIP 1) expression, e.g., liver cancer. In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer. 10 In a preferred embodiment the iRNA agent silences mutations in the PPM1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1D expression, e.g., breast cancer. In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS 15 expression, e.g., breast cancer. In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma. In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, 20 and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC). In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma. 25 In another preferred embodiment the iRNA agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum. In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with 30 chemotherapeutics. In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers. 209 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma In a preferred embodiment the iRNA agent silences mutations in the pRb tumor 5 suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC 1 expression, e.g., colon cancer. 10 In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer. In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder 15 characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers. In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias. 20 In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias. In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted 25 TEL/AML1 fusion gene expression, e.g., childhood acute leukemia. In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma. In another preferred embodiment the iRNA agent silences the TLS/FUS 1 fusion gene, 30 and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS 1 fusion gene expression, e.g., Myxoid liposarcoma. In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma. 210 WO 2004/094345 PCT/US2004/011822 In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia. In another aspect, the invention features, a method of treating a subject, e.g., a human, at 5 risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes: providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis; 10 administering the iRNA agent to a subject, thereby treating the subject. In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin. 15 In a preferred embodiment the iRNA agent silences the Fit-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis. In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. 20 Cancer and retinal neovascularization. In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization. In another aspect, the invention features a method of treating a subject infected with a 25 virus or at risk for or afflicted with a disorder or disease associated with a viral infection. The method includes: providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth; 30 administering the iRNA agent to a subject, preferably a human subject, thereby treating the subject. Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical 211 WO 2004/094345 PCT/US2004/011822 cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection. In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7. 5 In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced. The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS). 10 In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCR5, Gag, or Rev. In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD4 or Tsgl01. The invention also includes a method for treating patients infected by the Hepatitis B 15 Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma. In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another 20 preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail. In preferred embodiment the expression of a human gene that is required for HBV replication is reduced. The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV. 25 In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced. The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis In a preferred embodiment, the expression of a HCV gene is reduced. 30 In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced. The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis. 212 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced. In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced. 5 Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly. In a preferred embodiment, the expression of a RSV gene is reduced. In another 10 preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P. In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced. Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and 15 cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients. In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase pnrimase. 20 In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced. The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients. 25 In a preferred embodiment, the expression of a CMV gene is reduced. In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced. Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, 30 e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease. In a preferred embodiment, the expression of a EBV gene is reduced. In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced. 213 WO 2004/094345 PCT/US2004/011822 Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma. 5 In a preferred embodiment, the expression of a KSHV gene is reduced. In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced. The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal 10 leukoencephalopathy (PML). In a preferred embodiment, the expression of a JCV gene is reduced. In preferred embodiment the expression of a human gene that is required for JCV replication is reduced. Methods of the invention also provide for treating patients infected by the myxovirus or 15 at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza. In a preferred embodiment, the expression of a myxovirus gene is reduced. In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced. Methods of the invention also provide for treating patients infected by the rhinovirus or at 20 risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold. In a preferred embodiment, the expression of a rhinovirus gene is reduced. In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced. Methods of the invention also provide for treating patients infected by the coronavirus or 25 at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold. In a preferred embodiment, the expression of a coronavirus gene is reduced. In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced. Methods of the invention also provide for treating patients infected by the flavivirus West 30 Nile or at risk for or afflicted with a disorder mediated by West Nile Virus. In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5. 214 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced. Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this 5 virus, e.g., viral haemorrhagic fever or neurological disease. In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced. In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced. Methods of the invention also provide for treating patients infected by the Tick-borne 10 encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease. In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced. In a preferred embodiment the expression of a human gene that is required for Tick 15 borne encephalitis virus replication is reduced. Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease. In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is 20 reduced. In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced. The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever. 25 In a preferred embodiment, the expression of a dengue virus gene is reduced. In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced. Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis. 30 In a preferred embodiment, the expression of a SV40 gene is reduced. In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced. 215 WO 2004/094345 PCT/US2004/011822 The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy. In a preferred embodiment, the expression of a HTLV gene is reduced. In another 5 preferred embodiment the HTLV1 gene is the Tax transcriptional activator. In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced. Methods of the invention also provide for treating patients infected by the Moloney Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo 10 MuLV, e.g., T-cell leukemia. In a preferred embodiment, the expression of a Mo-MuLV gene is reduced. In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced. Methods of the invention also provide for treating patients infected by the 15 encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation. In a preferred embodiment, the expression of a EMCV gene is reduced. 20 In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced. The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles. In a preferred embodiment, the expression of a MV gene is reduced. 25 In a preferred embodiment the expression of a human gene that is required for MV replication is reduced. The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster). 30 In a preferred embodiment, the expression of a VZV gene is reduced. In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced. The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection. 216 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment, the expression of an adenovirus gene is reduced. In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced. The invention includes a method for treating patients infected by a yellow fever virus 5 (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection. In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes. 10 In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced. Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio. In a preferred embodiment, the expression of a poliovirus gene is reduced. 15 In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced. Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox In a preferred embodiment, the expression of a poxvirus gene is reduced. 20 In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced. In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes: providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA 25 is homologous to and can silence, e.g., by cleavage of a pathogen gene; administering the iRNA agent to a subject, prefereably a human subject, thereby treating the subject. The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production. 30 Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria. In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA1). 217 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced. The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers. 5 In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced. In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced. The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis. 10 In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced. In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced. The invention also includes methods for treating patients infected by the Mycobacterium 15 leprae, or a disease or disorder associated with this pathogen, e.g. leprosy. In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced. In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced. The invention also includes methods for treating patients infected by the bacteria 20 Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and museous membranes. In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced. In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced. 25 The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection. In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced. 30 In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced. The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever. 218 WO 2004/094345 PCT/US2004/011822 In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced. In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced. The invention also includes methods for treating patients infected by the bacteria 5 Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced. In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced. 10 The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced. In a preferred embodiment the expression of a human gene that is required for 15 Mycoplasma pneumoniae replication is reduced. In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder. The method includes: 20 providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response; administering the iRNA agent to a subject, thereby treating the subject. 25 In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplantated organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis. 30 In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty. In a prefered embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis. 219 WO 2004/094345 PCT/US2004/011822 In a prefered embodiment the disease or disorder is inflammation associated with an infection or injury. In a prefered embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic. 5 In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM. In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1). In particularly preferred embodiments the iRNA agent silences a component of the 10 complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase. In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL -2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYAll, CCR3. In other embodiments the iRNA agent silences GCSF, Grol, Gro2, Gro3, PF4, MIG, Pro 15 Platelet Basic Protein (PPBP), MIP-lI, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, 1-309. In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes: providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by 20 cleavage, a gene which mediates the processing of pain; administering the iRNA to a subject, thereby treating the subject. In particularly preferred embodiments the iRNA agent silences a component of an ion channel. 25 In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand. In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes: providing an iRNA agent which iRNA is homologous to and can silence, e.g., by 30 cleavage, a gene which mediates a neurological disease or disorder; administering the iRNA agent to a subject, thereby treating the subject. In a prefered embodiment the disease or disorder is Alzheimer's Disease or Parkinson Disease. 220 WO 2004/094345 PCT/US2004/011822 In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein. In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a 5 spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA7 or SCA8. In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJDl, CACNL1A4, SCA7, SCA8. The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease 10 state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential 15 for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected 20 allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH. E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR2A, 25 a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNA 30 synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor. Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes: optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell; 221 WO 2004/094345 PCT/US2004/011822 providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells; administerning the iRNA to the subject, thereby treating the disorder. 5 The invention also includes a iRNA agent disclosed herein, e.g, an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence 10 AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:28) is conserved between mouse lamin B 1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes. The invention also includes an iRNA agent disclosed herein, which can silence more than one gene. 15 ROUTE OF DELIVERY For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that 20 includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular. The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language 25 "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is 30 contemplated. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, 222 WO 2004/094345 PCT/US2004/011822 intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance targeting. For example, to 5 target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA. Formulations for topical administration may include transdermal patches, ointments, 10 lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. 15 In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined 20 with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added. Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Formulations for parenteral administration may include sterile aqueous solutions which 25 may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can 30 include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. 223 WO 2004/094345 PCT/US2004/011822 Topical Delivery For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., 5 modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. "Topical administration" refers to the delivery to a subject by 10 contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and 15 transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to 20 an underlying tissue. The term "skin," as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum comeum being at the surface of the 25 skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 trm and 0.2 mm thick, depending on its location on the body. Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings 30 and immunologically active cells. One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces 224 WO 2004/094345 PCT/US2004/011822 between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier. The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the 5 skin's permeability barrier, mechanisms other than normal osmosis must be used. Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the 10 epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum. Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic 15 conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition 20 disclosed herein for systemic and/or local therapy. In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the 25 cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites. 30 The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic 225 WO 2004/094345 PCT/US2004/011822 epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin. Pulmonary Delivery 5 For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger 10 iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar 15 region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs. Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose 20 devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be 25 mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament. The term "powder" means a composition that consists of finely dispersed solid particles 30 that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 pm in diameter preferably with a relatively uniform spheroidal shape 226 WO 2004/094345 PCT/US2004/011822 distribution. More preferably the diameter is less than about 7.5 /m and most preferably less than about 5.0 Am. Usually the particle size distribution is between about 0.1 ym and about 5 m in diameter, particularly about 0.3 Am to about 5 tm. The term "dry" means that the composition has a moisture content below about 10% by 5 weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol. The term "therapeutically effective amount" is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated 10 physiological response. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term "pharmaceutically acceptable carrier" means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs. 15 The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two. Bulking agents that are particularly valuable include compatible carbohydrates, 20 polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2 -hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, 25 raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred. Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, 30 phenylalanine, and the like. Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, 227 WO 2004/094345 PCT/US2004/011822 dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants. Oral or Nasal Delivery For ease of exposition the formulations, compositions and methods in this section are 5 discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma 10 levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily. In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of 15 the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible. The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 20 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae. 25 A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity. 30 Devices For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, 228 WO 2004/094345 PCT/US2004/011822 however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., 5 a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be 10 placed in the vasculature of the lung, heart, or leg. Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin. Other devices include artificial joints, e.g., hip joints, and other orthopedic implants. 15 In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics. Tissue, e.g., cells or organs, such as the kidney, can be treated with An iRNA agent, e.g., 20 a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject. ' The tissue can be autologous, allogeneic, or xenogeneic tissue. For example, tissue (e.g., 25 kidney) can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue, such as in the kidney. In another example, tissue containing hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation. Introduction of treated tissue, whether autologous or transplant, can be combined with 30 other therapies. In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate. 229 WO 2004/094345 PCT/US2004/011822 In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include 5 condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide. DOSAGE 10 In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, 15 optionally, (c) includes at least one 3' overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4 x 1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent 20 per kg of bodyweight. The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA, such as an RNA present in the kidney. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 25 mg/kg of body weight. In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. 30 In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent,. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, 230 WO 2004/094345 PCT/US2004/011822 e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty. In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., 5 a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 Ag to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 10 0.00001 mg per kg ofbodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once 15 for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed. 20 The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. 25 In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific. 30 In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a kidney disease, e.g., early stage renal disease, can be administered an iRNA agent specific for a target gene known to enhance the progression of the disease in conjunction with a drug known to inhibit activity of the target gene product. For example, a patient who has early stage renal disease can be treated with an iRNA 231 WO 2004/094345 PCT/US2004/011822 agent that targets an SGLT2 RNA, in conjunction with the small molecule phlorizin, which is known to block sodium-glucose cotransport and to subsequently reduce single nephron glomerular filtration rate. In another example, a patient being treated for a cancer of the kidney can be administered an iRNA agent specific for a target essential for tumor cell proliferation in 5 conjunction with a chemotherapy. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 gg to 100 g per kg of body weight (see US 6,107,094). 10 The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to 15 avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation. Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a 20 therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA 25 agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered. 30 Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages 232 WO 2004/094345 PCT/US2004/011822 may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding 5 endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human. The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of 10 ways. In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, 15 topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below. The invention provides methods, compositions, and kits, for rectal administration or 20 delivery of iRNA agents described herein. Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent 25 described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3' overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer. 30 The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication. The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also 233 WO 2004/094345 PCT/US2004/011822 by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose. Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can 5 be applied topically to a surface of the oral cavity. Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one o10 embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant. Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside 15 of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or 20 structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue. Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably 25 not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic 30 molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis. Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the 234 WO 2004/094345 PCT/US2004/011822 medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication. 5 Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue. iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery. Any of the iRNA agents described herein can be administered nasally. Nasal 10 administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered 15 dose of medication. The subject, or another person, can administer the medication. Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery. 20 An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom. The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned 25 above. Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent. A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which 30 can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein. 235 WO 2004/094345 PCT/US2004/011822 A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of 5 iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be o10 provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device. The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a 15 spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different 20 endogenous target RNAs. The method can include other features described herein. A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or 25 precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter 30 to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure 236 WO 2004/094345 PCT/US2004/011822 of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load). In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or 5 sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should 10 be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein. In another aspect, the invention features a method of administering a source of a double stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or 15 implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3' overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually 20 releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses. In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., 25 a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3' overhang 1-5 nt long. In one 30 embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome. In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The 237 WO 2004/094345 PCT/US2004/011822 microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which 5 can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1 o10 monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1- 1 0 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, 15 ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof. In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N amino acyl derivative of a beta-diketone or a mixture thereof. 20 In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof. In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti 25 inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., 30 a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a 238 WO 2004/094345 PCT/US2004/011822 cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric 5 material is a coating. The coating can be acetate phlithalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate. In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt 10 can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, laurie acid, and salts thereof. In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol. 15 In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate. In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides 20 long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3' overhang 1-5 nucleotides long. In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded 25 iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., 30 a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile 239 WO 2004/094345 PCT/US2004/011822 powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA 5 agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a 10 mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein. 15 In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage 20 form is a suppository. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. 25 In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., 30 a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof The microsphere can be formulated as a suspension, a powder, or an implantable solid. 240 WO 2004/094345 PCT/US2004/011822 In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, 5 including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide. In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected 10 from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose. 15 In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, 20 histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid. In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, c-lactalbumin, trypsinogen, and polyalanine. In one embodiment, the spray-dried iRNA agent composition includes particles having a 25 mass median diameter (MMAD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns. In certain other aspects, the invention provides kits that include a suitable container 30 containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the phannaceutical formulation may be provided in one container. Alternatively, it may be desirable 241 WO 2004/094345 PCT/US2004/011822 to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to 5 instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device. In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double 10 stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the 15 device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures. As used herein, the term "crystalline" describes a solid having the structure or 20 characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying. The invention is further illustrated by the following examples, which should not be 25 construed as further limiting. 242 WO 2004/094345 PCT/US2004/011822 EXAMPLES Examples 1-442 represent typical syntheses of the compounds delineated in Schemes 1 17; 101-116; and 1001-1005. 5 Scheme 1 a HO- NFmoc Si NFmoc i NH i HO .NO - 1 2 3 , Fmoc O-f" NH Fmoc Si N Si NH HO SI-O SSi 4 5 6 ai O[('Pr) 2 SiCI] 2 , Imidazole, DMF; ii TEA/MeCN 10 15 243 WO 2004/094345 PCTIUS2004/01 1822 Scheme 2 ' 0 0 N ' t 'CN O-R HO - 1 ,O-R '3 7a-I 8a-1 O O 0 I 1v -Si-O-si-0--- 'k / i-Si-. 0 O / "C Oa-I 9a" Z Me, Allyl or 0 Y p-cyanoethyl 0 0 d/-, N SO ""H COORt H H H H '~l~0 N<"... CODEt b n =1-17 H"., AcO H c n=1-17 d n =1-50(mfPEG) ~NI k h O -"- 0N 0' non /h I H e n =1-50 N H~ 5 244 WO 2004/094345 PCT/US2004/011822 a (i) (1) 1,1'-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 0 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylanmmonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 245 WO 2004/094345 PCTIUS2004/01 1822 Scheme 3 ' 0 0 siSi' 0 'P~ N% HO-b, NO-R Osi ** -so-O/ HO 6 i2a-I 13a iv Nw Ns - u "- N) N N 0 0 -*.p -Si-O N0 -Sl-. HO I V 14a-, -N , O vi-'~ 15a-1 ~- ~ -Z = Me, Allyl or NY 1-cyanoethyl 0 0 -Si-O-j-O-', 00 16a-i 0 H ~. COOEt H H * H R RA H H AcO HH aF f n b n1-17 cO H)~ d nl=-50 (mPEG) ~ NI k h >- N N e n=150) 5 246 WO 2004/094345 PCT/US2004/011822 a (i) (1) 1,1 '-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 Cl 2 ; (iv) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 247 WO 2004/094345 PCTIUS2004/01 1822 Scheme 4 6 l 7 (ip, NHI'5) 0 18 H -"ivN /I " \ -si- -S- -S1-, - Jkf p HO- 17 ( I 1-5)1 HO a- 0 Y HO Si- HO 0a I I V HO 20a. -Si-O-Si-O-' , I 9y /s -SI-O-si-O-, "RN -Si I I I 'P _ YO' 21a-1; Z=Me, Ally] or-/ 1-cyanoethyl 0 22a.1 H ".. COOEI H H t j~ ~ H n H fn11 H H n a n=1-17 H N d n =1-50(mPEG) h N N - N N a n 1-50N 5 248 WO 2004/094345 PCT/US2004/011822 a (i) FmocGly, DCC, DMAP / DMF; (ii) Et 3 N /MeCN; (iii) a. (1) 1,1' Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH; b. TBAF / THF; (iv) Benzhydryloxybis(trimethylsilyloxy)silyl-CI (BzHC1), diisopropylamine / CH 2 C1 2 ; (v) (a) Z = 5 Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= (3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (vi) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 o10 methylmorpholine, DMF, aminoalkyl solid support, rt. 249 WO 2004/094345 PCTIUS2004/01 1822 Scheme 5 ' 0 0 00 IHM. 0 A ,H I N 0R 010 0 00 1 Y 0"- N Y H 7p HdP 01vN N 0 R 00 H H Yi R a 0 1 H b n=1-17yl iiXZ' AcO gI n, O~ cn=1'17 H H

~

1 a( f2 I / eR='O H5 25 WO 2004/094345 PCT/US2004/011822 a (i) FmocGly, DCC, DMAP / DMF; (ii) Et 3 N /MeCN; (iii) a. (1) 1,1' Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH; b. TBAF / THF; (iv) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2

CI

2 ; (v) (a) Z = 5 Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (vi) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1l-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 o10 methylmorpholine, DMF, aminoalkyl solid support, rt. 251 WO 2004/094345 PCT/US2004/011822 Scheme 6 a /O NH /0 NINR HO -, N, R So C ' S S d 29a-I HO 30a-I O it - -Si-O-SS-- R -O- ,CO NR Si~ HO Si- O / 31a-I --- Poz V j32a-I; Z = Me, Allyl or O P-cyanoethyl -Si-O-S-O--C N-R 0 0o 'Si O' N S S 33a-I 0 H 00 RH H O O AcO a Ac H g h

\

0 o#oy CO x I o /n -q , b n= 1-17;q=0-4 FmocHN CO i X = Side chain of amino acid (Appropriately protected, Fmoc for O q CO lys, orn, benzyl for asp, glu etc [base labile protecting groups]) -n- q c n=1-17;q=0-4 FmocHN CO nCO j X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 - 16 lys, orn, benzyl for asp, glu etc [base labile protecting groups]) 0 X 0 FmocHN "CO FmocHNY N' N Yl en=l -16 X OH 0 x H k n = 0 - 20, X = Side chain of amino acid (Appropriately FnmocHN- N.,Vc fFmocHN N CO protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) f n= -16 5 252 WO 2004/094345 PCT/US2004/011822 a (i) RCOOH, DCC, DMAP / DMF; (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1l-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 253 WO 2004/094345 PCT/US2004/011822 Scheme 7 a Oi/-o - NH O-o,. NR HO-.. R Si Si O HO $ 6 34a-I 35a.I o 11 Siv -Si-O-Si- O-' . R - -S O-Si -O , R I -Si-_ HO I 36a-I N OZ 37a-I; Z= Me, Allyl or P3-cyanoethyl 0 O -S O Si O0, RO Si. O N / 38a-I 0 H 0 0 H H H AcO a AcO H O O(-O CO X O 0 b n 1-17;q =0 -4 FmocHN j CO I X = Side chain of amino acid (Appropriately protected, Fmoc for .O. OvqCO lys, orn, benzyl for asp, glu etc [base labile protecting groups]) n q c n 1-17;q =0-4 FmocHN~' CO CO j X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 - 16 lys, orn, benzyl for asp, glu etc [base labile protecting groups]) O X O FmocHN CO FmocHN N en=1-16 X X H k n = 0 -20, X = Side chain of amino acid (Appropriately FmocHN" N'- CO protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) fn=o-16 5 254 WO 2004/094345 PCT/US2004/011822 a (i) RCOOH, DCC, DMAP / DMF; (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 255 WO 2004/094345 PCT/US2004/011822 Scheme 8 a 0 0 O-o" NH 70- N NR HO-\ ),R SIH H H \ ~s,-O HO~ S 39a-g 40a-g -T< S HI I 42a-g; Z = Me, Allyl or p3-cyanoethyl -Si O-S -O- '- N N I I 'N N -Si- HO I 4243a-g O N' '~42a-g; Z =Me, Allyl or H 43a-g 0 H R= AcO omoCHN, an=0-16 dn=0-16 NH fn=0-16 FmocHN. EtOOC HN N bn=0-16 en=0-16 O 0 FCOCHN gn

=

0- 16;Y=H,Me,Etetc FaCOCHN. (< n cn=0-16 5a (i) (1) 1,1'-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2

C

2 ; (iv) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C12; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium o10 tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 256 WO 2004/094345 PCT/US2004/011822 Scheme 9 a 00 7 O N - -- ' N N N R R HON R 'I O O' O' O S 44a-g 45a-g I l i - - s- 0R -Si-O - N N KR S- O-- CN NR - - I- -) IN N *i H 0 H -Si O S HOH -Si- HdO 46a-g "-P'OZ vJ 47a-g; Z= Me, Allyl or O O-cyanoethyl -Si-O-Si-O .,R 'Si. 0O S I, N S 48a-g O H H R= AcO FmocHN NL)-5 an=0-16 dn=0-16 NH fn=0-16 FmocHN.- EtOOC YHN t-15 O~n HN N,( bn=0-16 en=0-16 O g n = 0- 16;Y= H, Me, Etete FaCOCHN n n cn=0-16 5 a (i) (1) 1,1 '-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) TBAF / THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2

C

2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium 10 tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 257 WO 2004/094345 PCT/US2004/011822 Scheme 10 a 0 O NO-R EtS- N -R Et3Si-O-N% O-R H O '--,C~ H- N OZO Ho" Hd "-Poz 8a-I 49a-I 5 60a-I;Z = Me, Allyl or 13-cyanoethyl O Et3Si-O N lO-R 0 51a-I 0 .aH COOEt H .aH R = H a R f Ho , COOEt b n 1-17 HH A% c n =1-17 N -~,H -~H 0 F d n= 1-50 (mPEG) h k -07- 0 04 Ii- NN N / N O -- nO IN en = 1-50 1 N 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1, 1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 258 WO 2004/094345 PCT/US2004/011822 Scheme 11a 0 O O EtzSi-O-7, N 1 O-R HO" N EtO-,Si-O-.. NNOR -N O R O O- H 0 HO HO N P'OZ 13a-I 52a-I 53a-.I; Z= Me, Allyl or iii -cyanoethyl O

E

t Si-O-,, PN O-R O 54a-I O .H '.. COOEt R= R H R = FR =R. k i a HH 0 fH H H. ' COOEt bn =1-17 H AcO c n=1-17 d n = 1-50 (mPEG) h k 00 0- 0 0 -o-:,,: -C'¢' 'O .. I NJ N N n

N

en = 1-50 5 a (i) Triethylsilyl chloride, imidazole / TI-IF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1 o10 yl)-1,1,3,3-tetramethyluroniuntetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 259 WO 2004/094345 PCT/US2004/011822 Scheme 12a OH 0 HO-,, P ,.N O'R Et Si-O-"',,N" - N O' R )~ PO 0 JP o 19a-I 55a-I vi EtSi-O- , ,, N N O ' O o N- Y 'R 0 O Et 3 Si-O-,, N O'R 50aI; Z = Me, Allyl or N j3-cyanoethyl 0 57a-I .H COOEt R= - H . H

R=

H H _H a Hf H N( o R..I . C O O Et b n=1-17 H AcOn c n=1-17 of d n = 1-50 (mPEG) h k 00- N n N e n=1-50 N 5a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, I H-tetrazole / CH 3 CN; (c) Z= P-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, NN-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1 1o yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 260 WO 2004/094345 PCT/US2004/011822 Scheme 13a O NO HO H H

HO-,,

N N- 0. 'O R OSI i, 0_ -He N N ' O R O 25a-I 58a.1 OvH EtSSi-O-,, AN O'R0O O59aI; Z = Me, Allyl or O p-cyanoethyl 60a-I .aH . " COOEt SH Hi C 0\ 0. NOO R bon = -17 H H dn = 1-50 (mPEG) h 04.O , "O, 0 NH e n =1-50N a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 Cl 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = [3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 261 WO 2004/094345 PCT/US2004/011822 Scheme 14 a EtoSi-O HO NREtzSi-O- NR ii Et3Si--

N'

R HH 61a-k N"K-P Oz 30a-kOZ iii 62a.k; Z = Me, Allyl or y P-cyanoethyl EtSi-O NRO 63a-k O O RH H AcO a AcO H H H g h -- 0 O CO x b n = 1-17; q= 0-4 FmocHN CO i X = Side chain of amino acid (Appropriately protected, Fmoc for .. Ovn CO lys, om, benzyl for asp, glu etc [base labile protecting groups]) n q c n=1-17;q=0-4 X FmocHN CO CO J X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0- 16 lys, orn, benzyl for asp, glu etc [base labile protecting groups]) FmocHN --CO FmocHN N en=1-16 X 0 X FmocHN k n = 0 - 20, X = Side chain of amino acid (Appropriately o N N-n"CO protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile SNH protecting groups]) fn=o- 16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH2C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= P-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 o10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 262 WO 2004/094345 PCT/US2004/011822 Scheme 15 a HO -,p -R 64a,0 EtzSi-O-t,. R HO-. NR Et3Si-O ,. ,N R

----

HO HO64Y 35a-k H 64a-k N-OZ 65a-k; Z= Me, Allyl or 0-cyanoethyl EtzSi-O-,,,..N' R O O N 0 H 66a-k O O R= H. H AcO a AcO H H H g h

\

0 O Co Ho x b n = 1-17;q=0-4 FmocHN CO i X= Side chain of amino acid (Appropriately protected, Fmoc for ( O,-)CO lys, orn, benzyl for asp, glu etc [base labile protecting groups]) --n q cn = 1-17; q=0-4 FmocHN CO nCO j X= Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 - 16 lys, om, benzyl for asp, glu etc [base labile protecting groups]) O X H O H FmocHN.J y ... FmocHN yi(A. 0 ,, e n =1-16 x H 0a x FmocHN N k n = 0 -20, X = Side chain of amino acid (Appropriately 1(N CO n protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) fn=o-16 5 (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / C11H2C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = P-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2 -(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 -methylmorpholine, DMF, aminoalkyl solid support, rt. 263 WO 2004/094345 PCT/US2004/011822 Scheme 16a 0 O O Etzi-O-o' N ) N R HO-,, N. R Et 3 Si-O-',, N NR HH pii H H H )w I H HO 'oz H40a-g 64a-g 65a-g; Z = Me, Allyl or iii -cyanoethyl O Et3Si--O-o'P )NNR 0 O O N S HH 0 66a-g R= AcO FmocHN N an =0-16 dn=0-16 NH fn=0-16 FmocHN EtOO YH bn-0-16 en=0-16 O F3COCHNg n = 0 -16; Y = H Me, Et etc cn=0-16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 264 WO 2004/094345 PCT/US2004/011822 Scheme 17a 0 O EtSi-O-.

2 Nk R HO N, R EtzSi-O- , __N ,R Hi H H &~H H Io HO 45a-g d67a-g OZ 68a-g; Z= Me, Allyl or iii 3-cyanoethyl Et3Si-O--P LN R N SO 0 69a-g H R= AcO FmocHN Nf an=O-16 dn=0-16 NH fn=0-16 FmocHN EtOOCY H R : -- 5 CR :5 YHN. N ,( f bn=0-16 en=-16n FCOCHN g n = 0- 16;Y=H, Me, Etetc cn=0-16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH3CN; (c) Z = 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 265 WO 2004/094345 PCTIUS2004/01 1822 Scheme lol' 0 0 Bz0 BzO 1 R HO- R NHN 0- N 0' C AcO"'ZN) AcO"-* HO 101 102a.1 103a.1 0 0 0 0. il1 0 1v-jOS -*b -S-i-O-SI-O 'k R iv--w-S0-.0 N N01 / 104a.1-I-,O 01Y v~ , ~ I 105a-1; Z = Me, Allyl or 0 10, -cyanoethyl 106a-I 0 H COOEt HH H~ HH R a 0 RR 0H ". CODEt H b n=117 1~ AcO nn-1 d n 1 1-50 (mnPEG) h -_YN N -'( _ e n =1-50 I - N 5 266 WO 2004/094345 PCT/US2004/011822 a (i) (1) 1,1'-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) LiOH / THF-H 2 0; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 267 WO 2004/094345 PCTIUS2004/01 1822 Scheme 102' 0 0 AcO>-II A 0 .O ~ HO/ 107 1O8a-! 109a.1 N~ N I N (Y I I N lila1-; Z=Me, Ally! or V 3-cyanoethyl -s 0 -Si-0-Si-o"; 112a-IO0 H H H a 0 f A aH N h, COOR H nN c n =1-17 *.?H eH d n 1 -50 (mPEG) h h/

-

0 ~~- -~ 0NN 4 j H o no 1-0 1 \ 5 268 WO 2004/094345 PCT/US2004/011822 a (i) (1) 1,1'-Carbonyldiimidazole (CDI), DMAP / TiF; (2) R-OH, (ii) LiOH / THF-H1 2 0; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-Cl (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,NV-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 269 WO 2004/094345 PCTIUS2004/01 1822 Scheme 103' O-1YO-PNHFmnoc 0 BzO-,>"NH Bzo- N YE)-N 0' R 107 Aco 1 3 a-I (p 1-15) AuG11a 0N OYO-,N)L-: R O O yO%0. HO-,,. ~N H0 y) iv j I HO l~ HO 116a-j ~ ~'I - vi N -N O -k o-L R N Y 0 y N.~ . -si-O-Si-O-,,, N NH -Si 0 1 117a-I;Z= Me. Allyl or 0 llfla-I 0-ryanoethyl 'H "... COQEt H H o R= - A Hq N.&N a 0 H f 0 H COGEt "RO H b n=1-17HH AcOg c nl=-17 d n 1-5O(mnPEG) I 0 N (N -- N NJ 0o no N_\ e n =1-50 - N 5 270 WO 2004/094345 PCT/US2004/011822 a (i) FmocGly, DCC, DMAP / DMF; (ii) Et 3 N /MeCN; (iii) a. (1) 1,1' Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH; b. LiOH / THF-H 2 0; (iv) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH2C1 2 ; (v) (a) Z= 5 Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= f3 cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (vi) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 10 methylmorpholine, DMF, aminoalkyl solid support, rt. 271 WO 2004/094345 PCTIUS2004/01 1822 Scheme 104' 0 1 '0'NHFmoc; BzO' NH BzO N pO-p.~N JNO-R Acd 101 11 9a-I (p 1-15) AdO 120a-I 0z I O~N)o O. lk R 0 HO N H v0 oy( PQLN 0 ' R HO11a IH S!2 H1 122a Cii 4i 122a IH k0 -S o 0 0"14pi 0 N SN 123a-I; Z = Me, Allyl or 0 P-cyanoeihyl 124a-1 COOEt H_ * - H H AO H c n= 1-17 d n =1-50 (mPEG) h ~ 0 0 H CN 5 272 WO 2004/094345 PCT/US2004/011822 a (i) FmocGly, DCC, DMAP / DMF; (ii) Et 3 N/MeCN; (iii) a. (1) 1,1' Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH; b. LiOH / THF-H 2 0; (iv) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 Cl 2 ; (v) (a) Z = 5 Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamnidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (vi) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1, 1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 10 methylmorpholine, DMF, aminoalkyl solid support, rt. 273 WO 2004/094345 PCT/US2004/011822 Scheme 105 a BzO R BzO NH BzO HO N AcO, )w AcdO Hd sIS H' 127a- -Si 0 O 128a-I; Z = Me, Allyl or R I,-cyanoethyl N O 10 On CO AcO X 0 b n= 1-17;aq =0-4 O=MNlCo c n1-17;q0-4 FmocHN CO C O J X = Side chain of amino acid (Appropriately protected, Fmoc for dn = 0 -16 lys, or, benzyl for asp, glu etc [base labile protecting groups]) O X O mocHN "CO FmocHN YJI

H

N) 5 en=1-16 X O X H k n = 0 - 20, X = Side chain of amino acid (Appropriately N CmOcnN " protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) fn=o-16 5 274 WO 2004/094345 PCT/US2004/011822 a (i) RCOOH, DCC, DMAP / DMF; (ii) LiOH / THF-H1 2 0; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2

C

2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z= allyl: 5 diisopropylanie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= (3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 275 WO 2004/094345 PCT/US2004/011822 Scheme 106a Bo R R BzO-N.. H BzO-,, N HO-,, AcO AcO 106 130a-I 130a. 0 S I O I 1 0 R J!~ -S-O-Si-O-',,., N iv II F-s !o-sj-o-,,N HO 131a-I -i 0 I-P O R132a-I; Z= Me, Ally or I I -cyanoethyl -Si-O-Si--o-,, N ,0 P 0 0 133a-O H O' " 0 .,\H '. Ho R= ,, H H . • ', AcO a HI -o CO AcO H g h b n=1-17; q=0-4 0 FmocHN CO CO iX = Side chain of amino acid (Appropriately protected, Fmoc for - 17;q lys, orn, benzyl for asp, glu etc [base labile protecting groups]) c n=1-17;q=0-4 x FmocHN 'CO "( C O j X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 -16 lys, om, benzyl for asp, glu etc [base labile protecting groups]) FmocHN - CO FmocHNy N N en=1-16 X H X Fmoc H k n = 0 -20, X = Side chain of amino acid (Appropriately N Y Vn CO protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) f n = o - 16 5 276 WO 2004/094345 PCT/US2004/011822 a (i) RCOOH, DCC, DMAP / DMF; (ii) LiOH / THF-H 2 0; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: 5 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3 cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH3CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(lH-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 10 277 WO 2004/094345 PCT/US2004/011822 Scheme 107 a H H BzO-,.N O N ' R O N' R NH BzO-,, N HO-, N AcOU 106 Ac H 134a.g 135a-g H O O N , -O O o H N ' -si o- ON -sIo s vHOZ -N I 136a-g / NPZ YO 137a-g; Z = Me, Allyl or R 0I-cyanoethyl -Si-O-Si-O-,, N 'Si O ON 0 H 138a-g R= AcO FmocHN Nf, 3 an=0-16 dn=0-16 NH fn= 0-16 FmocHNO EtO YH bn=O-16 en=0-16 0 g n = 0- 16; Y= H, Me, Et etc

F

3

COCHN-.

on=0-16 5 a (i) (1) 1,1 '-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) LiOH / THF THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2

C

CI

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium 10 tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1l-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 278 WO 2004/094345 PCT/US2004/011822 Scheme 108 a H H BzO ON'R yN'R H BzO N HO N AcO"' 101 AcO HO 1H 139a.g 140a-g H HJ O O N'R O N, R S-S-O-Si O N -- Siv I N Si- HO .. S HO 141a-g -Si 0 NP'O \).: OL '142a-g; Z = Me, Allyl or H JI-cyanoethyl O N'R I O -Si O- Si- O N I I 143a.g R = AcOf FmocHNYNO 5 an=0-16 dn=0-16 NH fn=0-16 FmocHN - EtOOC4$ H bn=0-16 en=0-16 Y O FaCOCHN g n = 0 -16; Y = H, Me, Etetc cn=0-16 5 a (i) (1) 1,1'-Carbonyldiimidazole (CDI), DMAP / THF; (2) R-OH, (ii) LiOH / THF THF; (iii) Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; (iv) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium 10 tetrazolide, CH 3 CN, rt; (v) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 279 WO 2004/094345 PCT/US2004/011822 Scheme 109 a o O O'R HO NOR EtSi-0 Et 3 Si--O N NOR 0 HO- N"o'Ri EtAi-O0 -- R i . HO,,,. N,. 0-- IN,. . _ , HOHOO 103a-I 144a- OZ 0 , ii 145a-1; Z = Me, Allyl or S R 3-cyanoethyl Et3Si-O _ O 0 N S 0 1 4 6 aiH mH,. COOEt H H .H H H H H a 0 R f Hnh' i,... , Caoor H COOt -nO H H b n 1-17 . AcO R no . c n =1-17 d n = 1-50(mPEG) h ~ -~N NJN e n 1-50 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2

C

2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2 -(1H-benzotriazole-1 lo yl)-1,1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 280 WO 2004/094345 PCTIUS2004/01 1822 Scheme Il10' R 00 0 HO2) 0k R t 3 0i-0-S. IL..N 107a-I 147a-I NPOZ H ~ 148a-1; Z =Me. Allyl or 0 y 01 R -cyanoethyl EtSi-O-4j' 0 149a-IH ".. COQOt H H .% - H H A HR a H N p4,, -COOEt b n=1-17 AcO c n =1-17H H *-0 k d n = -5o (mPEG) h 0 -- 0 0 -J0 N N n -O 4 C

N

e n=1-50 I a (i) Triethylsilyl chloride, imidazole / THE; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiainidite, 1 H-tetrazole / CT{ 2

C

2 ; (b) Z = allyl: dilsopropylamie, (allyloxy)bis(diisopropylamino)phospine, 1 H-tetrazole / CH 3 CN; (c) Z =f3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, NN-diisopropylanu'onium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichioroethane, DMAP, (C 2 H5) 3 N, rt; (2) 2 -(1H-benzotriazole-1 10o yl)- 1,1 , 3

,

3 -tetramnethyluronjumtetrafluoroborate (TBTUJ), 4 -methylmorpholine, DMF, aininoalcyl solid support, rt. 281 WO 2004/094345 PCT/US2004/011822 Scheme 111 a 0 O O O O-RN OLOR O NORO N 0,R EtSi-0-,N Ho HO- , . N 0-. PH O- - ,Et3Si-O-,. 0 H -1H 10a- 'OZ HO HO N-Poz 115a-I 151a-I; Z = Me, Allyl or j}-cyaneethyl O Oy- P N-, R Et3Si-O--," N- H 0 ON 0 152a-I . - COOEt H H .H H H R= H H a H 1 , COOEt NO H b n =1-17 AcO nO c n 1-17 _1 d n = 1-50(mPEG) h -o--x -o 5 -0 ,NH O nO N N en =1-50 1 N 5a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = P-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, NN-diisopropylammonium tetrazolide, CH 3 GN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(lH-benzotriazole-l 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 282 WO 2004/094345 PCT/US2004/011822 Scheme 112a 0 0 O O N O-R O -N -IOR O N OR EtzSi-O N H HO Et 3 Si-O-EtNi H Hd N-loz 121a-I 153a-I - 154a-I; Z = Me, Allyl or iii P-cyanoethyl EtNSi-O

-

O N-0 0 H 155a-I .... COOEt H . H R= \H a 0 HI COOEt H H b n=1-17 AcO c n = 1-17 dn = 1-50 (mPEG) h -.O O- O N- HN Sn = 1-50N N 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 o10 yl)- 1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 283 WO 2004/094345 PCT/US2004/011822 Scheme 113 a R REt3Si-O N R R HO Et 3 Si-O ii HO156a-k OZ 126a-k iii 157a-k; Z = Me, Allyl or P-cyanoethyl 0 158a-k 0 0 t,,°,, 0 mHH AcO R= a H H x0 AcO H h b n=1-17;q=0-4 O FmacHN CO , nO vqCO i X = Side chain of amino acid (Appropriately protected, Fmoc for c n= 1-17;q=0-4 lys, cm, benzyl for asp, glu etc [base labile protecting groups]) c n=1-17;q=0-4 x FmocHN CO C O j X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 - 16 lys, om, benzyl for asp, glu etc [base labile protecting groups]) FmocHN CCO FmocHN ,% N YJ en=1-16 X x FmocHN k n = 0 - 20, X = Side chain of amino acid (Appropriately iN "n'Co protected, Fmoc for lys, om, benzyl for asp, glu etc [base labile NH protecting groups]) f n = o-16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= f3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2-(1H-benzotriazole-1 o10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rft. 284 WO 2004/094345 PCT/US2004/011822 Scheme 114 a R R Et 3 Si-O-,,.N HO-,,. N Et 3 Si-O-, N I I HO HO oz 130a-k 19a-k OZ I 160a-k; Z = Me, Allyl or Et3So N i O-cyanoethyl Et3Si-O-"- - - N 0 O N S 161a-k O 0 ,, H H AcO a H H --. O CO AcO H g h O - Jn - q b n=1-17;q=0-4 O FmocHN CO O CO iX = Side chain of amino acid (Appropriately protected, Fmoc for c n= 1-17;q 0-4 lys, orn, benzyl for asp, glu etc [base labile protecting groups]) c n=1-17;qz0-4 x FmocHN CO " C O j X = Side chain of amino acid (Appropriately protected, Fmoc for d n = 0 - 16 lys, orn, benzyl for asp, glu etc [base labile protecting groups]) O X O FmocHN ... FmocHN N " co" Fm; H N ,,N e n=lI - 16 X 0 X FmocHN YHI n k n = 0 -20, X = Side chain of amino acid (Appropriately N CO protected, Fmoc for lys, orn, benzyl for asp, glu etc [base labile NH protecting groups]) fn =n -16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= P3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 285 WO 2004/094345 PCT/US2004/011822 Scheme 115a H H H O NR O Y N' R O N-R EtaSi-0-oN HO-,Ezi- -, Ho-,, NEt 3 Si-O---,, N i HO HO 162ag NPOZ 135a-g lii 163a-g; Z= Me, Allyl or H p-cyanoethyl

O

0 R EItaSi-O-,,. .__ 0 H 164a.g R= AcO n FmocHN N an=O-16 dn=0-16 NH fn=0-16

FMOCHN,()&

7 EtOOC4&J',- 5 C YHY~ bn=0-16 en=0-16 F3COCHN g n = 0- 16; Y= H, Me, Et etc cn=0-16 5 a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= P-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 286 WO 2004/094345 PCT/US2004/011822 Scheme 116a H HO N Et 3 Si-O N N0 HO HO NP 140a-g OZ 165a-g 166a-g; Z = Me, Allyl or iii p3-cyanoethyl H Et 3 -O N R 0 M o H R 167a-gFmocHN _- cO(s FmocHNYt.(- 5 an=O-16 dn=0-16 NH fn=0-1B FmocHNO 5 EtOOC Y H ff,-( YHN. Nf bn=0_16 en=0-16 0 O

F

3 COCHN i g n =0 -16; Y= H, Me,Etetc cn=0-16 5a (i) Triethylsilyl chloride, imidazole / THF; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = j3-cyanoethyl: 2 cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH3CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1 10 yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4-methylmorpholine, DMF, aminoalkyl solid support, rt. 287 WO 2004/094345 PCTIUS2004/01 1822 Scheme 1001a R' ~Ri HO' R"-Si-O BO R"- R"B 0 N, ..o CACE HO OH HO OACE 1001 a-s lOO2a-s R', R' = O-SiMe; R' =O-CH(C 6

H

5

)

2 &1 x03 X =allyl, methyl or Et3Si R S~' 0 -cyanoethyl Et 3 i-F B B W, 0 HO OACE s N 0 OACE 10052-s H 1004a-s RRR Ethyl R', R" = O-S!Me.3; R" = O-CH(CcH 5

)

2 Et 3 Si-O B ACE = bis(2-acetoxyethoxy)mnethy1 p 10 OACE &1006a-s X = allyl, methyl or p-cyanoethyl B NH N(CH 3 )Q N(CH 3

)

2

N(CH

3 )Q N(CH 3

)

2 NHO 0 N~~0 N\ -NNN "'" INI N K'N N 'N NK N N N N N N NNAN 0 0 0 NHCONHCH 2 COOq" HN: IN 0 HNJI C 0 HN) 0) N)-N 0-'N N N 0-O -- N N -N N Hi h I I i k 0CON ~ , NHQ CHN NHO NHQ 0 HNN \ HN 0 HN N C\ H ~ O N N N j O)N N N NMeS - N N H M ~ H nl HSOCNHCOOCH3 NHBzQ1 N HN N N N ON NHn H r 5 288 WO 2004/094345 PCT/US2004/011822 a(i) a. Tris(2-acetoxyethoxy)orthoformate, pyridniump-toluenesulfonate, 4-(tert butyldimethylsilyloxy)-3-penten-2-one, dioxane, rt; b. TMEDA-HF, MeCN; c. Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 Cl 2 ; (ii) (a) Z= 5 Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH2C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z = 3 cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1-yl)-1,1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 10 methylmorpholine, DMF, aminoalkyl solid support, rt. (iv) a. Tris(2 acetoxyethoxy)orthoformate, pyridnium p-toluenesulfonate, 4-(tert-butyldimethylsilyloxy)-3 penten-2-one, dioxane, rt; b. TMEDA-HF, MeCN; c.Triethylsilyl chloride, imidazole / THF 289 WO 2004/094345 PCTIUS2004/01 1822 Scheme 1002' R'-Si-O 1 HO FT'-Si-O A N-. 0 OACE HO OH HO CACE P 09 1007a.u 1OO8a-u R', R' = O-SiMe 3 ; R' O-CH(C 6

H

5

)

2 0.. xO~ ivli X = allyl, methyl or R' P-cyanoethyl Et 3 Si-O BR'T-Si-0 HO OACE CG N JL,,o OACE l0lla-u H 0l1l1a-u R', R', R'V = Ethyl R', R'= O-SiMe 3 ; R" O-CH(C 6

H

5

)

2 EtSi-O B ACE = bis(2-acetoxyethoxy)methyl N,. 0 OACE & 1012..u X . alyl, methyl or 1-cyanoethyl B NHBz NHBz 0 0 0 0 0 F3 ' N NH HN) OAc HN' CAC N O R s NLS N --- SO0- N S N o -~ b d e O" f O N p HN'P HN HNp HNP OX SI)N 0" N S-)'N 0 N d' g i h ~ i ON jk NHBz N I~ NHBz 0 NHBz NHBz NH11z NN -N' N- N' \NY~ N HBz NHBzq r BzHNI'N -N BZHN~ N 5 290 WO 2004/094345 PCT/US2004/011822 a(i) a. O[('Pr) 2 SiC1] 2 , imidazole, DMF; b. Tris(2-acetoxyethoxy)orthoformnate, pyridnium p-toluenesulfonate, 4 -(tert-butyldimethylsilyloxy)-3-penten-2-one, dioxane, rt; c. TMEDA-HF, MeCN; d. Benzhydryloxybis(trimethylsilyloxy)silyl-Cl (BzHC1), diisopropylamine / CH2C1 2 ; (ii) 5 (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1l-yl)-1,1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 o10 methylmorpholine, DMF, aminoalkyl solid support, rt. (iv) a. O[(iPr) 2 SiC1] 2 , imidazole, DMF; b. Tris(2-acetoxyethoxy)orthoformate, pyridniump-toluenesulfonate, 4-(tert butyldimethylsilyloxy)-3-penten-2-one, dioxane, rt; c. TMEDA-IF, MeCN; d.Triethylsilyl chloride, imidazole / THF 291 WO 2004/094345 PCTIUS2004/01 1822 Scheme 1003 a RI' R-Si-O HO R"-Si-O 'a " B N)4 .O Y 1013a-j Y =H or OH 1014a-j R', R" = O-SiMe 3 ; R" =O-CH(C 6

H

5

)

2 , 0, 1015a-I 1iv YHor OAEX=al] methyl or Et 3 Si-O B II R-SI-O B 1017a-j H 0 1016a-j R', R", R" Ethyl R', R'= O-SiMe.3; R" O-CH(CrH 5

)

2 Eti-0B, ACE = bis(2-acetoxyethoxy)methyl N, .-O Y 6P 1018.a-i X = elyl, methyl or 0-cyanoethyl B NHBz NHQ 0 0 N N- r> HN HN N O1N KN N ' N NH b C d e f 0 00NHO NHQ N NH H N N N N N Jr 5 292 WO 2004/094345 PCT/US2004/011822 a(i) For Y = OH, a. Tris(2-acetoxyethoxy)orthoformate, pyridniump-toluenesulfonate, 4 (tert-butyldimethylsilyloxy)-3-penten-2-one, dioxane, rt; b. TMEDA-HF, MeCN; c. Benzhydryloxybis(trimethylsilyloxy)silyl-C1 (BzHC1), diisopropylamine / CH 2 C1 2 ; For Y = H: 5 Benzhydryloxybis(trimethylsilyloxy)silyl-Cl (BzHC1), diisopropylamine / CH 2 C1 2 ; (ii) (a) Z= Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH2C1 2 ; (b) Z= allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 3 cyanoethyl: 2 -eyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 10 2-(1H-benzotriazole-1-yl)-1,1, 3

,

3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. (iv) a. For Y = OH, a. Tris(2 acetoxyethlioxy)orthoformate, pyridnium p-toluenesulfonate, 4 -(tert-butyldimethylsilyloxy)-3 penten-2-one, dioxane, rt; b. TMEDA-HF, MeCN; c. Benzhydryloxybis(trimethylsilyloxy)silyl Cl (BzHC1), diisopropylamine / CH 2

C

2 ; For Y = H: Benzhydryloxybis(trimethylsilyloxy)silyl 15 Cl (BzHC1), diisopropylamine / CH2 C 2 ; 293 WO 2004/094345 PCT/US2004/011822 Scheme 1004a AcO pAc AcO oAc AcO .OAc

F

3 COCHN""

F

3 COCHN,

F

3 COCHN,, O

NHCOCF

3 O O O NHCOCFa O NHCOCF 3 0 0 00 0 0 H' NH R'SI oN HO R"-SI-O R' N HoR" Y 1021a HO HO N pO 1021b 1019 1020a R', R", R" = Ethyl or X = allyl, methyl or 1020b R', R" = O-SiMe 3 ; R"' = X P-cyanoethyl

O-CH(C

6 H5) 2 i AcO OAc

F

3

COCHNI,

O

NHCOCF

3 R' HN R"-Si-O N R"' 0 1 022a S N 0 1022b H O R', R", R"'= Ethyl or R', R" = O-SiMe 3 ; R'" = O-CH(C 6 Hs) 2 5 a(i) For 8a: Triethylsilyl chloride, imidazole / THF; For 8b: Benzhydryloxybis(trimethylsilyloxy)silyl-Cl (BzHC1), diisopropylamine / CH 2

C

2 ; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: 10 diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= p cyanoethyl: 2 -cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2

H

5

)

3 N, rt; (2) 2 -(1H-benzotriazole-1-yl)-1,1,3, 3 -tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. 15 294 WO 2004/094345 PCTIUS2004/01 1822 Scheme 1005a HO R'Si-O aBR'-Si-(:, BR w N, 0 HO HO P,105a-z 1023a-z 1024a-z R', R" = O-SiMe,; R" O-CH(C6H 5

)

2 ~ 025 c 1023a1-cl 1024a1-ci ii X 2ally-tyio Et 3 Si-O iv R"Slr 0cyanoethyl M R HO 1026a-z S N 01026a-z 1026a1 -cl O H 0 01026al-cl R' " "= Ethyl R, R" = O-SiMe3; R" = O-CH(CcH 5

)

2 Et 2 Si-O ACE = bis(2-acetoxyethoxy)methyl P 1027a-z X = allyl, methyl or 1-cyancethyl 0,1027a1-cl B 0 N0

H

2 N H 2 N H 2 N H- 2 N HN N\ NN N N 0 0 0 0 0 ~ N N N ' (( bi c Nd N e Jf -Jg

NO

2

NO

2

NO

2 N0 2 NHQ 0 0 2 NO; HN N I N-\ N CNH NO N N_ N N N(CON2 (Ny N02 N ~

SCONH

2 N Np -N N O N F N-': NN I N, NHQ 0-)- I F N 0 N 0 Y iil ibi ci 5 295 WO 2004/094345 PCT/US2004/011822 a(i) Benzhydryloxybis(trimethylsilyloxy)silyl-Cl (BzHC1), diisopropylamine / CH 2 C1 2 ; (ii) (a) Z = Me: methyl tetraisopropyl phosphorodiamidite, 1 H-tetrazole / CH 2 C1 2 ; (b) Z = allyl: diisopropylamie, (allyloxy)bis(diisopropylamino)phosphine, 1 H-tetrazole / CH 3 CN; (c) Z= 0 5 cyanoethyl: 2-cyanoethyltetraisopropylphosphorodiamidite, N,N-diisopropylammonium tetrazolide, CH 3 CN, rt; (iii) (1) succinic anhydride, 1,2-dichloroethane, DMAP, (C 2 Hs) 3 N, rt; (2) 2-(1H-benzotriazole-1l-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU), 4 methylmorpholine, DMF, aminoalkyl solid support, rt. (iv) Triethylsilyl chloride, imidazole / TIHF 10 Example 1 Compound 2 (Scheme 1): The diol 1 is prepared as reported in the literature (Filichev and Pedersen, Tetrahedron, 2001, 57, 9163-68). 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.9 mmol) is added into a solution of compound 1 (1 mmol) and imidazole (3 mmol) in DMF to 15 obtain compound 2 (Evans et al., Tetrahedron, 2000, 56, 3053-62). Example 2 Compound 3 (Scheme 1): Compound 2 is stirred with triethylamine in acetonitrile to obtain compound 3 (Filichev and Pedersen, Tetrahedron, 2001, 57, 9163-68). Example 3 20 Compound 6 (Scheme 1): The diol 4 is prepared according to the literature reports by Filichev and Pedersen (Tetrahedron, 2001, 57, 9163-68). Cyclic silylether is prepared from compound 4 according to literature procedure (Evans et al., Tetrahedron, 2000, 56, 3053-62). Treatment of compound 5 with TEA in acetonitrile yields compound 6. Example 4 25 Compound 7a (Scheme 2): Compound 3 (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Cholesterol (1 nmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 7a (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). 30 Example 5 Compound 8a (Scheme 2): A solution of compound 7a in THF is treated with TBAF for lh at ambient temperature to yield compound 8a (Evans et al., Tetrahedron, 2000, 56, 3053-62). Example 6 296 WO 2004/094345 PCT/US2004/011822 Compound 9a (Scheme 2): The desired silyl ether 9a is obtained by the treatment of compound 8a with benzhydryloxybis(trimethylsilyloxy)silyl chloride (BzH-C1) in the presence of diisopropylamine as reported by Chui et al. (J. Org. Chem., 2002, 67, 8847-54). Example 7 5 Compound 10a (Z = Me, Scheme 2): Treatment of compound 9a with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in

CH

2 C1 2 yields compound 10a (Chui et al. J Org. Chem., 2002, 67, 8847-54). Example 8 Compound 10a (Z = allyl, Scheme 2): To a solution of compound 9a (1 mmol) in 10 acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mnunol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the desired phosphoramidite 10a (Hayakawa et al., J Am. Chem. Soc., 1990, 112, 1691-96). Example 9 15 Compound 10Oa (Z= P-cyanoethyl, Scheme 2): The desired phosphoramidite 10a is prepared from compound 9a and P-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 10 20 Solid support 11a (Scheme 2): Compound 9a (1 mmol) is mixed with succinic anhydride (2 nmmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethne (0.9 mL) is added into the mixture, and the mixture is stirred at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The 25 organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4 methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid 30 support (118.9 pmol/g, particle size 80/120, mean pore diameter 569 A) is added into the clear solution, and the mixture is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized 297 WO 2004/094345 PCT/US2004/011822 sites on the SOLID SUPPORT are capped with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) and allows to shake on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The final loading capacity of 85a is determined after capping 5 (Prakash et al., J Org. Chem. 2002, 67, 357-369). Example 11 Compound 10b (Z = Me, n = 15, Scheme 2): The phosphoramidite 10b is prepared from compound 3 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 7. 10 Example 12 Compound 10b (Z = allyl, n = 15, Scheme 2): The phosphoramidite 10b is prepared from compound 3 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 8. Example 13 15 Compound 10b (Z= P-cyanoethyl, n = 15, Scheme 2): The phosphoramidite 10b is prepared from compound 3 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 9. Example 14 20 Solid support 11b (n = 15, Scheme 2): The solid support 11b is prepared from compound 9b as reported in Example 10. Compound 9b is obtained from compound 3 and 1,3 bis-O-(hexadecyl)glycerol as reported in Examples 4, 5 and 6. Example 15 Compound 10c (Z = Me, n = 15, Scheme 2): The phosphoramidite 10c is prepared from 25 compound 3 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 7. Example 16 Compound 10c (Z= allyl, n = 15, Scheme 2): The phosphoramidite 10c is prepared from compound 3 and hexadecylglycerol according to the procedures described in Examples 4, 30 5, 6 and 8. Example 17 Compound 10c (Z = P-cyanoethyl, n = 15, Scheme 2): The phosphoramidite 10c is prepared from compound 3 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 9. 298 WO 2004/094345 PCT/US2004/011822 Example 18 Solid support 11c (n = 15, Scheme 2): The solid support 11c is prepared from compound 9c as reported in Example 10. Compound 9c is obtained from compound 3 and hexadecylglycerol as reported in Examples 4, 5 and 6. 5 Example 19 Compound 10d (Z = Me, Scheme 2): The phosphoramidite 10d is prepared from compound 3 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. Example 20 10 Compound 10d (Z= allyl, Scheme 2): The phosphoramidite 10d is prepared from compound 3 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. 299 WO 2004/094345 PCT/US2004/011822 Example 21 Compound 10d (Z= P-cyanoethyl, Scheme 2): The phosphoramidite 10d is prepared from compound 3 and desired Me-O-PEG according to the procedures described in Examples 4, 5 5, 6 and9. Example 22 Solid support 11 d (Scheme 2): The solid support 11d is prepared from compound 9d as reported in Example 10. Compound 9d is obtained from compound 3 and desired Me-O-PEG as reported in Examples 4, 5 and 6. 10 Example 23 Compound 10e (Z = Me, Scheme 2): The phosphoramidite 10e is prepared from compound 3 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. Example 23 15 Compound 10e (Z= allyl, Scheme 2): The phosphoramidite 10e is prepared from compound 3 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. Example 24 Compound 10e (Z= 3-cyanoethyl, Scheme 2): The phosphoramidite 10e is prepared 20 from compound 3 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. Example 25 Solid support lIe (Scheme 2): The solid support 11le is prepared from compound 9e as reported in Example 10. Compound 9e is obtained from compound 3 and desired branched Me 25 O-PEG as reported in Examples 4, 5 and 6. Example 26 Compound 10f (Z = Me, Scheme 2): The phosphoramidite 10f is prepared from compound 3 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 7. 30 300 WO 2004/094345 PCT/US2004/011822 Example 27 Compound 10f (Z = allyl, Scheme 2): The phosphoramidite 10f is prepared from compound 3 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 5 and 8. Example 28 Compound 10f (Z = 3-cyanoethyl, Scheme 2): The phosphoramidite 10f is prepared from compound 3 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and9. 10 Example 29 Solid support 11f (Scheme 2): The solid support 11f is prepared from compound 9f as reported in Example 10. Compound 9f is obtained from compound 3 and dihydrotestosteron as reported in Examples 4, 5 and 6. Example 30 15 Compound 10g (Z = Me, Scheme 2): The phosphoramidite 10g is prepared from compound 3 and borneol according to the procedures described in Examples 4, 5, 6 and 7. Example 31 Compound 10g (Z = allyl, Scheme 2): The phosphoramidite 10g is prepared from compound 3 and bomeol according to the procedures described in Examples 4, 5, 6 and 8. 20 Example 32 Compound 10g (Z = P-cyanoethyl, Scheme 2): The phosphoramidite 10g is prepared from compound 3 and borneol according to the procedures described in Examples 4, 5, 6 and 9. Example 33 Solid support 11g (Scheme 2): The solid support 11g is prepared from compound 9g as 25 reported in Example 10. Compound 9g is obtained from compound 3 and borneol as reported in Examples 4, 5 and 6. 301 WO 2004/094345 PCT/US2004/011822 Example 34 Compound 10h (Z = Me, Scheme 2): The phosphoramidite 10h is prepared from compound 3 and menthol according to the procedures described in Examples 4, 5, 6 and 7. 5 Example 35 Compound 10h (Z = allyl, Scheme 2): The phosphoramidite 10h is prepared from compound 3 and menthol according to the procedures described in Examples 4, 5, 6 and 8 Example 36 Compound 10h (Z= O-cyanoethyl, Scheme 2): The phosphoramidite 10h is prepared 10 from compound 3 and menthol according to the procedures described in Examples 4, 5, 6 and 9. Example 37 Solid support 11h (Scheme 2): The solid support llh is prepared from compound 9h as reported in Example 10. Compound 9h is obtained from compound 3 and menthol as reported in Examples 4, 5 and 6. 15 Example 38 Compound 10i (Z= Me, Scheme 2): The phosphoramidite 10i is prepared from compound 3 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and7. Example 39 20 Compound 10i (Z= allyl, Scheme 2): The phosphoramidite 10i is prepared from compound 3 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. Example 40 Compound 10i (Z = 0-cyanoethyl, Scheme 2): The phosphoramidite 10i is prepared 25 from compound 3 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. 302 WO 2004/094345 PCT/US2004/011822 Example 41 Solid support 11i (Scheme 2): The solid support 11i is prepared from compound 9i as reported in Example 10. Compound 9i is obtained from compound 3 and cholenic acid as 5 reported in Examples 4, 5 and 6. Example 42 Compound 10j (Z = Me, Scheme 2): The phosphoramidite 10j is prepared from compound 3 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 7. 10 Example 43 Compound 10j (Z = allyl, Scheme 2): The phosphoramidite 10j is prepared from compound 3 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. Example 44 15 Compound 10j (Z = f3-cyanoethyl, Scheme 2): The phosphoramidite 10j is prepared from compound 3 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. Example 45 Solid support llj (Scheme 2): The solid support 1lj is prepared from compound 9j as 20 reported in Example 10. Compound 9j is obtained from compound 3 and lithocholic acid ethyl ester as reported in Examples 4, 5 and 6. Example 46 Compound 10k (Z= Me, Scheme 2): The phosphoramidite 10k is prepared from compound 3 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 7. 25 Example 47 Compound 10k (Z= allyl, Scheme 2): The phosphoramidite 10k is prepared from compound 3 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 8. 303 WO 2004/094345 PCT/US2004/011822 Example 48 Compound 10k (Z = 3-cyanoethyl, Scheme 2): The phosphoramidite 10k is prepared from compound 3 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 5 9. Example 49 Solid support 11k (Scheme 2): The solid support Ilk is prepared from compound 9k as reported in Example 10. Compound 9k is obtained from compound 3 and Vitamin E as reported in Examples 4, 5 and 6. 10 Example 50 Compound 101 (Z= Me, Scheme 2): The phosphoramidite 101 is prepared from compound 3 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 7. Example 51 15 Compound 101 (Z = allyl, Scheme 2): The phosphoramidite 101 is prepared from compound 3 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 8. Example 52 Compound 101 (Z = P-cyanoethyl, Scheme 2): The phosphoramidite 101 is prepared 20 from compound 3 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 9. Example 53 Solid support 111 (Scheme 2): The solid support 111 is prepared from compound 91 as reported in Example 10. Compound 91 is obtained from compound 3 and Hoechst 33258 - PEG 25 conjugate as reported in Examples 4, 5 and 6. Example 54 Compound 15a (Z = Me, Scheme 3): The phosphoramidite 15a is prepared from compound 6 and cholesterol according to the procedures described in Examples 4, 5, 6 and 7. 30 304 WO 2004/094345 PCT/US2004/011822 Example 55 Compound 15a (Z = allyl, n = 15, Scheme 3): The phosphoramidite 15a is prepared from compound 6 and cholesterol according to the procedures described in Examples 4, 5, 6 and 5 8. Example 56 Compound 15a (Z = P-cyanoethyl, Scheme 3): The phosphoramidite 15a is prepared from compound 6 and cholesterol according to the procedures described in Examples 4, 5, 6 and 9. 10 Example 57 Solid support 16a (Scheme 3): The solid support 16a is prepared from compound 14a as reported in Example 10. Compound 14a is obtained from compound 6 and 1,3-bis-O (hexadecyl)glycerol as reported in Examples 4, 5 and 6. Example 58 15 Compound 15b (Z = Me, n = 15, Scheme 3): The phosphoramidite 15b is prepared from compound 6 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 7. Example 59 Compound 15b (Z= allyl, n = 15, Scheme 3): The phosphoramidite 15b is prepared 20 from compound 6 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 8. Example 60 Compound 15b (Z = P-cyanoethyl, n = 15, Scheme 3): The phosphoramidite 15b is prepared from compound 6 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures 25 described in Examples 4, 5, 6 and 9. Example 61 Solid support 16b (n = 15, Scheme 3): The solid support 16b is prepared from compound 9b as reported in Example 10. Compound 14b is obtained from compound 6 and 1,3 bis-O-(hexadecyl)glycerol as reported in Examples 4, 5 and 6. 30 305 WO 2004/094345 PCT/US2004/011822 Example 62 Compound 15c (Z = Me, n = 15, Scheme 3): The phosphoramidite 15c is prepared from compound 6 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 5 and 7. Example 63 Compound 15c (Z = allyl, n = 15, Scheme 3): The phosphoramidite 15c is prepared from compound 6 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 8. 10 Example 64 Compound 15c (Z = 3-cyanoethyl, n = 15, Scheme 3): The phosphoramidite 15c is prepared from compound 6 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 9. Example 65 15 Solid support 16c (n = 15, Scheme 3): The solid support 16c is prepared from compound 14c as reported in Example 10. Compound 14c is obtained from compound 6 and hexadecylglycerol as reported in Examples 4, 5 and 6. Example 66 Compound 15d (Z = Me, Scheme 3): The phosphoramidite 15d is prepared from 20 compound 6 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. Example 67 Compound 15d (Z = allyl, Scheme 3): The phosphoramidite 15d is prepared from compound 6 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 25 and 8. Example 68 Compound 15d (Z= 1-cyanoethyl, Scheme 3): The phosphoramidite 15d is prepared from compound 6 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. 30 306 WO 2004/094345 PCT/US2004/011822 Example 69 Solid support 16d (Scheme 3): The solid support 16d is prepared from compound 14d as reported in Example 10. Compound 14d is obtained from compound 6 and desired Me-O-PEG 5 as reported in Examples 4, 5 and 6. Example 70 Compound 15e (Z = Me, Scheme 3): The phosphoramidite 15e is prepared from compound 6 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. 10 Example 71 Compound 15e (Z= allyl, Scheme 3): The phosphoramidite 15e is prepared from compound 6 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. Example 72 15 Compound 15e (Z = P-cyanoethyl, Scheme 3): The phosphoramidite 15e is prepared from compound 6 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. Example 73 Solid support 16e (Scheme 3): The solid support 16e is prepared from compound 14e as 20 reported in Example 10. Compound 14e is obtained from compound 6 and desired branched Me O-PEG as reported in Examples 4, 5 and 6. Example 74 Compound 15f (Z = Me, Scheme 3): The phosphoramidite 15f is prepared from compound 6 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 25 and 7. Example 75 Compound 15f (Z= allyl, Scheme 3): The phosphoramidite 15f is prepared from compound 6 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 8. 30 307 WO 2004/094345 PCT/US2004/011822 Example 76 Compound 15f (Z= 3-cyanoethyl, Scheme 3): The phosphoramidite 15f is prepared from compound 6 and dihydrotestosteron according to the procedures described in Examples 4, 5 5, 6 and9. Example 77 Solid support 16f (Scheme 3): The solid support 16f is prepared from compound 14f as reported in Example 10. Compound 14f is obtained from compound 6 and dihydrotestosteron as reported in Examples 4, 5 and 6. 10 Example 78 Compound 15g (Z = Me, Scheme 3): The phosphoramidite 15g is prepared from compound 6 and bomrneol according to the procedures described in Examples 4, 5, 6 and 7. Example 79 Compound 15g (Z = allyl, Scheme 3): The phosphoramidite 15g is prepared from 15 compound 6 and borneol according to the procedures described in Examples 4, 5, 6 and 8. Example 80 Compound 15g (Z = 1-cyanoethyl, Scheme 3): The phosphoramidite 15g is prepared from compound 6 and borneol according to the procedures described in Examples 4, 5, 6 and 9. Example 81 20 Solid support 16g (Scheme 3): The solid support 16g is prepared from compound 14g as reported in Example 10. Compound 14g is obtained from compound 6 and borneol as reported in Examples 4, 5 and 6. Example 82 Compound 15h (Z = Me, Scheme 3): The phosphoramidite 15h is prepared from 25 compound 6 and menthol according to the procedures described in Examples 4, 5, 6 and 7. 308 WO 2004/094345 PCT/US2004/011822 Example 83 Compound 15h (Z= allyl, Scheme 3): The phosphoramidite 15h is prepared from compound 6 and menthol according to the procedures described in Examples 4, 5, 6 and 8. 5 Example 84 Compound 15h (Z= -cyanoethyl, Scheme 3): The phosphoramidite 15h is prepared from compound 6 and menthol according to the procedures described in Examples 4, 5, 6 and 9. Example 85 Solid support 16h (Scheme 3): The solid support 16h is prepared from compound 14h 10 as reported in Example 10. Compound 14h is obtained from compound 6 and menthol as reported in Examples 4, 5 and 6. Example 86 Compound 16i (Z = Me, Scheme 3): The phosphoramidite 16i is prepared from compound 6 and cholenic acid ethyl ester according to the procedures described in Examples 4, 15 5,6 and7. Example 87 Compound 15i (Z= allyl, Scheme 3): The phosphoramidite 15i is prepared from compound 6 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. 20 Example 88 Compound 15i (Z = P3-cyanoethyl, Scheme 3): The phosphoramidite 15i is prepared from compound 6 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. Example 89 25 Solid support 16i (Scheme 3): The solid support 16i is prepared from compound 14i as reported in Example 10. Compound 14i is obtained from compound 6 and cholenic acid as reported in Examples 4, 5 and 6. 309 WO 2004/094345 PCT/US2004/011822 Example 90 Compound 15j (Z= Me, Scheme 3): The phosphoramidite 15j is prepared from compound 6 and lithocholic acid ethyl ester according to the procedures described in Examples 5 4, 5, 6 and 7. Example 91 Compound 15j (Z = allyl, Scheme 3): The phosphoramidite 15j is prepared from compound 6 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. 10 Example 92 Compound 15j (Z = O-cyanoethyl, Scheme 3): The phosphoramidite 15j is prepared from compound 6 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. Example 93 15 Solid support 16j (Scheme 3): The solid support 16j is prepared from compound 14j as reported in Example 10. Compound 14j is obtained from compound 6 and lithocholic acid ethyl ester as reported in Examples 4, 5 and 6. Example 94 Compound 15k (Z = Me, Scheme 3): The phosphoramidite 15k is prepared from 20 compound 6 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 7. Example 95 Compound 15k (Z= allyl, Scheme 3): The phosphoramidite 15k is prepared from compound 6 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 8. Example 96 25 Compound 15k (Z = 0-cyanoethyl, Scheme 3): The phosphoramidite 15k is prepared from compound 6 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 9. 310 WO 2004/094345 PCT/US2004/011822 Example 97 Solid support 16k (Scheme 3): The solid support 16k is prepared from compound 14k as reported in Example 10. Compound 14k is obtained from compound 6 and Vitamin E as 5 reported in Examples 4, 5 and 6. Example 98 Compound 151 (Z = Me, Scheme 3): The phosphoramidite 151 is prepared from compound 6 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 7. 10 Example 99 Compound 151 (Z = allyl, Scheme 3): The phosphoramidite 151 is prepared from compound 6 and Hoechst 33258 -PEG conjugate according to the procedures described in Examples 4, 5, 6 and 8. Example 100 15 Compound 151 (Z = j3-cyanoethyl, Scheme 3): The phosphoramidite 151 is prepared from compound 6 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 9. Example 101 Solid support 111 (Scheme 3): The solid support 161 is prepared from compound 141 as 20 reported in Example 10. Compound 141 is obtained from compound 6 and Hoechst 33258 -PEG conjugate as reported in Examples 4, 5 and 6. Example 102 Compound 17 (p = 1, Scheme 4): Compound 6 (1 nmmol, Scheme 1) is coupled to Fmoc Gly in the presence of DCC. A solution of Fmoc-Gly (1 mmol), DhbhOH (1.5 mmol) and DCC 25 (1.1 mmol) in dry DMF (2 mL) is stirred at ambient temperature for 2 h after which compound 6 (1.2 rmmol) is added into the stirring reaction mixture. The reaction is stirred for 8 h. DCU is removed by filtration and DMF is removed under vacuum. The residue is suspended in ethyl acetate and is subjected bicarbonate wash to remove any unreacted acid followed by water wash and subsequently with KHSO 4 solution to remove excess amine. Standard workup and 30 chromatographic purification is followed to obtain compound 17. Example 103 Compound 18 (Scheme 4): Compound 17 is stirred in DMF-piperidine (9:1) for 30 min to yield compound 18. 311 WO 2004/094345 PCT/US2004/011822 Example 104 Compound 21a (Z = Me, Scheme 4): The phosphoramidite 21a is prepared from compound 18 and cholesterol according to the procedures described in Examples 4, 5, 6 and 7. Example 105 5 Compound 21a (Z= allyl, Scheme 4): The phosphoramidite 21a is prepared from compound 18 and cholesterol according to the procedures described in Examples 4, 5, 6 and 8. Example 106 Compound 21a (Z = P3-cyanoethyl, Scheme 4): The phosphoramidite 21a is prepared from compound 18 and cholesterol according to the procedures described in Examples 4, 5, 6 O10 and 8. Example 107 Solid support 22a (Scheme 4): The solid support 22a is prepared from compound 20a as reported in Example 10. Compound 20a is obtained from compound 18 and 1,3-bis-O (hexadecyl)glycerol as reported in Examples 4, 5 and 6. 15 Example 108 Compound 21b (Z= Me, n = 15, Scheme 4): The phosphoramidite 21b is prepared from compound 18 and 1, 3 -bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 7. Example 109 20 Compound 21b (Z= allyl, n = 15, Scheme 4): The phosphoramidite 21b is prepared from compound 18 and 1, 3 -bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 8. 312 WO 2004/094345 PCT/US2004/011822 Example 110 Compound 21b (Z = 0-cyanoethyl, n = 15, Scheme 4): The phosphoramidite 21b is prepared from compound 18 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures 5 described in Examples 4, 5, 6 and 9. Example 111 Solid support 22b (n = 15, Scheme 4): The solid support 22b is prepared from compound 20b as reported in Example 10. Compound 20b is obtained from compound 18 and 1,3-bis-O-(hexadecyl)glycerol as reported in Examples 4, 5 and 6. 10 Example 112 Compound 21c (Z= Me, n = 15, Scheme 4): The phosphoramidite 21c is prepared from compound 18 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 7. Example 113 15 Compound 21c (Z = allyl, n = 15, Scheme 4): The phosphoramidite 21c is prepared from compound 18 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 8. Example 114 Compound 21c (Z = P3-cyanoethyl, n = 15, Scheme 4): The phosphoramidite 21c is 20 prepared from compound 18 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 9. Example 115 Solid support 22c (n = 15, Scheme 4): The solid support 22c is prepared from compound 20c as reported in Example 10. Compound 20c is obtained from compound 18 and 25 hexadecylglycerol as reported in Examples 4, 5 and 6. Example 116 Compound 21d (Z = Me, Scheme 4): The phosphoramidite 21d is prepared from compound 18 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. 30 313 WO 2004/094345 PCT/US2004/011822 Example 117 Compound 21d (Z = allyl, Scheme 4): The phosphoramidite 21d is prepared from compound 18 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 5 and 8. Example 118 Compound 21d (Z= 03-cyanoethyl, Scheme 4): The phosphoramidite 21d is prepared from compound 18 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. 10 Example 119 Solid support 22d (Scheme 4): The solid support 22d is prepared from compound 20d as reported in Example 10. Compound 20d is obtained from compound 18 and desired Me-O PEG as reported in Examples 4, 5 and 6. Example 120 15 Compound 21e (Z = Me, Scheme 4): The phosphoramidite 21e is prepared from compound 18 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. Example 121 Compound 21e (Z = allyl, Scheme 4): The phosphoramidite 21e is prepared from 20 compound 18 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. Example 122 Compound 21e (Z= 3-cyanoethyl, Scheme 4): The phosphoramidite 21e is prepared from compound 18 and desired branched Me-O-PEG according to the procedures described in 25 Examples 4, 5, 6 and 9. Example 123 Solid support 22e (Scheme 4): The solid support 22e is prepared from compound 20e as reported in Example 10. Compound 20e is obtained from compound 18 and desired branched Me-O-PEG as reported in Examples 4, 5 and 6. 30 314 WO 2004/094345 PCT/US2004/011822 Example 124 Compound 21f (Z = Me, Scheme 4): The phosphoramidite 21f is prepared from compound 18 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 5 and 7. Example 125 Compound 21f (Z = allyl, Scheme 4): The phosphoramidite 21f is prepared from compound 18 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 8. 10 Example 126 Compound 21f (Z = P-cyanoethyl, Scheme 4): The phosphoramidite 21f is prepared from compound 18 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 9. Example 127 15 Solid support 22f (Scheme 4): The solid support 22f is prepared from compound 20f as reported in Example 10. Compound 20f is obtained from compound 18 and dihydrotestosteron as reported in Examples 4, 5 and 6. Example 128 Compound 21g (Z = Me, Scheme 4): The phosphoramidite 21g is prepared from 20 compound 18 and borneol according to the procedures described in Examples 4, 5, 6 and 7. Example 129 Compound 21g (Z = allyl, Scheme 4): The phosphoramidite 21g is prepared from compound 18 and bomrneol according to the procedures described in Examples 4, 5, 6 and 8. Example 130 25 Compound 21g (Z = P-cyanoethyl, Scheme 4): The phosphoramidite 21g is prepared from compound 18 and borneol according to the procedures described in Examples 4, 5, 6 and 9. 315 WO 2004/094345 PCT/US2004/011822 Example 131 Solid support 22g (Scheme 4): The solid support 22g is prepared from compound 20g as reported in Example 10. Compound 20g is obtained from compound 18 and bomrneol as reported 5 in Examples 4, 5 and 6. Example 132 Compound 21h (Z= Me, Scheme 4): The phosphoramidite 21h is prepared from compound 18 and menthol according to the procedures described in Examples 4, 5, 6 and 7. Example 133 10 Compound 21h (Z = allyl, Scheme 4): The phosphoramidite 21h is prepared from compound 18 and menthol according to the procedures described in Examples 4, 5, 6 and 8. Example 134 Compound 21h (Z = (3-cyanoethyl, Scheme 4): The phosphoramidite 21h is prepared from compound 18 and menthol according to the procedures described in Examples 4, 5, 6 and 9. 15 Example 135 Solid support 22h (Scheme 4): The solid support 22h is prepared from compound 20h as reported in Example 10. Compound 20h is obtained from compound 18 and menthol as reported in Examples 4, 5 and 6. Example 136 20 Compound 21i (Z= Me, Scheme 4): The phosphoramidite 21i is prepared from compound 18 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 7. Example 137 Compound 21i (Z= allyl, Scheme 4): The phosphoramidite 21i is prepared from 25 compound 18 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. 316 WO 2004/094345 PCT/US2004/011822 Example 138 Compound 21i (Z = P3-cyanoethyl, Scheme 4): The phosphoramidite 21i is prepared from compound 18 and cholenic acid ethyl ester according to the procedures described in 5 Examples 4, 5, 6 and 9. Example 139 Solid support 22i (Scheme 4): The solid support 22i is prepared from compound 20i as reported in Example 10. Compound 20i is obtained from compound 18 and cholenic acid as reported in Examples 4, 5 and 6. 10 Example 140 Compound 21j (Z = Me, Scheme 4): The phosphoramidite 21j is prepared from compound 18 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 7. Example 141 15 Compound 21j (Z = allyl, Scheme 4): The phosphoramidite 21j is prepared from compound 18 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. Example 142 Compound 21j (Z= -cyanoethyl, Scheme 4): The phosphoramidite 21j is prepared 20 from compound 18 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. Example 143 Solid support 22j (Scheme 4): The solid support 22j is prepared from compound 20j as reported in Example 10. Compound 20] is obtained from compound 18 and lithocholic acid ethyl 25 ester as reported in Examples 4, 5 and 6. Example 144 Compound 21k (Z = Me, Scheme 4): The phosphoramidite 21k is prepared from compound 18 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 7. 317 WO 2004/094345 PCT/US2004/011822 Example 145 Compound 21k (Z = allyl, Scheme 4): The phosphoramidite 21k is prepared from compound 18 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 8. 5 Example 146 Compound 21k (Z= P-cyanoethyl, Scheme 4): The phosphoramidite 21k is prepared from compound 18 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 9. Example 147 10 Solid support 22k (Scheme 4): The solid support 22k is prepared from compound 20k as reported in Example 10. Compound 20k is obtained from compound 18 and Vitamin E as reported in Examples 4, 5 and 6. Example 148 Compound 211 (Z = Me, Scheme 4): The phosphoramidite 211 is prepared from 15 compound 18 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 7. Example 149 Compound 211 (Z = allyl, Scheme 4): The phosphoramidite 211 is prepared from compound 18 and Hoechst 33258 - PEG conjugate according to the procedures described in 20 Examples 4, 5, 6 and 8. Example 150 Compound 211 (Z = -cyanoethyl, Scheme 4). The phosphoramidite 211 is prepared from compound 18 and Hoechst 33258 -PEG conjugate according to the procedures described in Examples 4, 5, 6 and 9. 25 Example 151 Solid support 221 (Scheme 4): The solid support 221 is prepared from compound 201 as reported in Example 10. Compound 201 is obtained from compound 18 and Hoechst 33258 PEG conjugate as reported in Examples 4, 5 and 6. 318 WO 2004/094345 PCT/US2004/011822 Example 152 Compound 23 (Scheme 5): Compound 3 (1 mmol, Scheme 1) is coupled to Fmoc-Gly in the presence of DCC. A solution of Fmoc-Gly (1 mmol), DhbhOH (1.5 mmol) and DCC (1.1 5 nmmol) in dry DMF (2 mL) is stirred at ambient temperature for 2 h after which compound 3 (1.2 mmol) is added into the stirring reaction mixture. The reaction is stirred for 8 h. DCU is removed by filtration and DMF is removed under vacuum. The residue is suspended in ethyl acetate and is subjected to bicarbonate wash to remove any unreacted acid followed by water wash and subsequently with KHSO 4 solution to remove excess amine. Standard workup and 10 chromatographic purification is followed to obtain compound 23. Example 153 Compound 24 (Scheme 5): Compound 23 is stirred in DMF-piperidine (9:1) for 30 min to yield compound 24. Example 154 15 Compound 27a (Z= Me, Scheme 5): The phosphoramidite 27a is prepared from compound 24 and cholesterol according to the procedures described in Examples 4, 5, 6 and 7. Example 155 Compound 27a (Z = allyl, Scheme 5): The phosphoramidite 27a is prepared from compound 24 and cholesterol according to the procedures described in Examples 4, 5, 6 and 8. 20 Example 156 Compound 27a (Z = j-cyanoethyl, Scheme 5): The phosphoramidite 27a is prepared from compound 24 and cholesterol according to the procedures described in Examples 4, 5, 6 and 8. Example 157 25 Solid support 28a (Scheme 5): The solid support 28a is prepared from compound 26a as reported in Example 10. Compound 26a is obtained from compound 24 and 1,3-bis-O (hexadecyl)glycerol as reported in Examples 4, 5 and 6. Example 158 30 Compound 27b (Z= Me, n = 15, Scheme 5): The phosphoramidite 27b is prepared from compound 24 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 7. 319 WO 2004/094345 PCT/US2004/011822 Example 159 Compound 27b (Z = allyl, n = 15, Scheme 5): The phosphorarnidite 27b is prepared from compound 24 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 8. 5 Example 160 Compound 27b (Z = P-cyanoethyl, n = 15, Scheme 5): The phosphoramidite 27b is prepared from compound 24 and 1,3-bis-O-(hexadecyl)glycerol according to the procedures described in Examples 4, 5, 6 and 9. Example 161 10 Solid support 28b (n = 15, Scheme 5): The solid support 28b is prepared from compound 26b as reported in Example 10. Compound 26b is obtained from compound 24 and 1,3-bis-O-(hexadecyl)glycerol as reported in Examples 4, 5 and 6. Example 162 Compound 27c (Z = Me, n = 15, Scheme 5): The phosphoramidite 27e is prepared from 15 compound 24 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 7. Example 163 Compound 27c (Z = allyl, n = 15, Scheme 5): The phosphoramidite 27c is prepared from compound 24 and hexadecylglycerol according to the procedures described in Examples 4, 20 5, 6 and 8. Example 164 Compound 27c (Z = P3-cyanoethyl, n = 15, Scheme 5): The phosphoramidite 27c is prepared from compound 24 and hexadecylglycerol according to the procedures described in Examples 4, 5, 6 and 9. 25 320 WO 2004/094345 PCT/US2004/011822 Example 165 Solid support 28c (n = 15, Scheme 5): The solid support 28c is prepared from compound 26c as reported in Example 10. Compound 26c is obtained from compound 24 and hexadecylglycerol as reported in Examples 4, 5 and 6. 5 Example 166 Compound 27d (Z = Me, Scheme 5): The phosphoramidite 27d is prepared from compound 24 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. Example 167 10 Compound 27d (Z= allyl, Scheme 5): The phosphoramidite 27d is prepared from compound 24 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. Example 168 Compound 27d (Z = P-cyanoethyl, Scheme 5): The phosphoramidite 27d is prepared 15 from compound 24 and desired Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. Example 169 Solid support 28d (Scheme 5): The solid support 28d is prepared from compound 26d as reported in Example 10. Compound 26d is obtained from compound 24 and desired Me-O 20 PEG as reported in Examples 4, 5 and 6. Example 170 Compound 27e (Z = Me, Scheme 5): The phosphoramidite 27e is prepared from compound 24 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 7. 25 Example 171 Compound 21e (Z = allyl, Scheme 5): The phosphoramidite 27e is prepared from compound 24 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 8. 321 WO 2004/094345 PCT/US2004/011822 Example 172 Compound 27e (Z = 0-cyanoethyl, Scheme 5): The phosphoramidite 27e is prepared from compound 24 and desired branched Me-O-PEG according to the procedures described in Examples 4, 5, 6 and 9. 5 Example 173 Solid support 28e (Scheme 5): The solid support 28e is prepared from compound 26e as reported in Example 10. Compound 26e is obtained from compound 24 and desired branched Me-O-PEG as reported in Examples 4, 5 and 6. Example 174 10 Compound 27f (Z = Me, Scheme 5): The phosphoramidite 27f is prepared from compound 28 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 7. Example 175 Compound 27f (Z = allyl, Scheme 5): The phosphoramidite 27f is prepared from 15 compound 24 and dihydrotestosteron according to the procedures described in Examples 4, 5, 6 and 8. Example 176 Compound 27f (Z = P3-cyanoethyl, Scheme 5): The phosphoramidite 27f is prepared from compound 24 and dihydrotestosteron according to the procedures described in Examples 4, 20 5, 6 and 9. Example 177 Solid support 28f (Scheme 5): The solid support 28f is prepared from compound 26f as reported in Example 10. Compound 26f is obtained from compound 24 and dihydrotestosteron as reported in Examples 4, 5 and 6. 25 Example 178 Compound 27g (Z = Me, Scheme 5): The phosphoramidite 27g is prepared from compound 24 and bomrneol according to the procedures described in Examples 4, 5, 6 and 7. 322 WO 2004/094345 PCT/US2004/011822 Example 179 Compound 27g (Z = allyl, Scheme 5): The phosphoramidite 27g is prepared from compound 24 and borneol according to the procedures described in Examples 4, 5, 6 and 8. Example 180 5 Compound 27g (Z = 1-cyanoethyl, Scheme 5): The phosphoramidite 27g is prepared from compound 24 and borneol according to the procedures described in Examples 4, 5, 6 and 9. Example 181 Solid support 28g (Scheme 5): The solid support 28g is prepared from compound 26g as reported in Example 10. Compound 26g is obtained from compound 24 and borneol as reported 10 in Examples 4, 5 and 6. Example 182 Compound 27h (Z = Me, Scheme 5): The phosphoramidite 27h is prepared from compound 24 and menthol according to the procedures described in Examples 4, 5, 6 and 7. Example 183 15 Compound 27h (Z = allyl, Scheme 5): The phosphoramidite 27h is prepared from compound 24 and menthol according to the procedures described in Examples 4, 5, 6 and 8. Example 184 Compound 27h (Z= 1-cyanoethyl, Scheme 5): The phosphoramidite 27h is prepared from compound 24 and menthol according to the procedures described in Examples 4, 5, 6 and 9. 20 Example 185 Solid support 28h (Scheme 5): The solid support 28h is prepared from compound 26h as reported in Example 10. Compound 26h is obtained from compound 24 and menthol as reported in Examples 4, 5 and 6. Example 186 25 Compound 27i (Z= Me, Scheme 5): The phosphoramidite 27i is prepared from compound 24 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 7. Example 187 Compound 27i (Z= allyl, Scheme 5): The phosphoramidite 27i is prepared from 30 compound 24 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. 323 WO 2004/094345 PCT/US2004/011822 Example 188 Compound 27i (Z= -cyanoethyl, Scheme 5): The phosphoramidite 27i is prepared from compound 24 and cholenic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 9. 5 Example 189 Solid support 28i (Scheme 5): The solid support 28i is prepared from compound 26i as reported in Example 10. Compound 26i is obtained from compound 24 and cholenic acid as reported in Examples 4, 5 and 6. Example 190 10 Compound 27j (Z = Me, Scheme 5): The phosphoramidite 27j is prepared from compound 28 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 7. Example 191 Compound 27j (Z= allyl, Scheme 5): The phosphoramidite 27j is prepared from 15 compound 24 and lithocholic acid ethyl ester according to the procedures described in Examples 4, 5, 6 and 8. Example 192 Compound 27j (Z = 3-cyanoethyl, Scheme 5): The phosphoramidite 27j is prepared from compound 24 and lithocholic acid ethyl ester according to the procedures described in 20 Examples 4, 5, 6 and 9. Example 193 Solid support 28j (Scheme 5): The solid support 28j is prepared from compound 26j as reported in Example 10. Compound 26j is obtained from compound 28 and lithocholic acid ethyl ester as reported in Examples 4, 5 and 6. 25 Example 194 Compound 27k (Z= Me, Scheme 5): The phosphoramidite 27k is prepared from compound 24 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 7. Example 195 Compound 27k (Z = allyl, Scheme 5): The phosphoramidite 27k is prepared from 30 compound 24 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 8. Example 196 Compound 27k (Z = 3-cyanoethyl, Scheme 5): The phosphoramidite 27k is prepared from compound 24 and Vitamin E according to the procedures described in Examples 4, 5, 6 and 9. 324 WO 2004/094345 PCT/US2004/011822 Example 197 Solid support 28k (Scheme 5): The solid support 28k is prepared from compound 26k as reported in Example 10. Compound 26k is obtained from compound 24 and Vitamin E as reported in Examples 4, 5 and 6. 5 Example 198 Compound 271 (Z = Me, Scheme 5): The phosphoramidite 271 is prepared from compound 24 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 7. Example 199 10 Compound 271 (Z = allyl, Scheme 5): The phosphoramidite 271 is prepared from compound 24 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 8. Example 200 Compound 271 (Z = P3-cyanoethyl, Scheme 5): The phosphoramidite 271 is prepared 15 from compound 24 and Hoechst 33258 - PEG conjugate according to the procedures described in Examples 4, 5, 6 and 9. Example 201 Solid support 281 (Scheme 5): The solid support 281 is prepared from compound 261 as reported in Example 10. Compound 261 is obtained from compound 24 and Hoechst 33258 20 PEG conjugate as reported in Examples 4, 5 and 6. Example 202 Compound 29a (Scheme 6): Compound 3 is coupled to 0 3 -(acetyl)cholenic acid in the presence of DCC and DMAP in DMF. The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29a. 25 Example 203 Compound 32a (Z= Me, Scheme 6): The phosphoramidite 32a is prepared from compound 29a according to the procedures described in Examples 5, 6 and 7. Example 204 Compound 32a (Z = allyl, Scheme 6): The phosphoramidite 32a is prepared from 30 compound 29a according to the procedures described in Examples 5, 6 and 8. Example 205 Compound 32a (Z= -cyanoethyl, Scheme 6): The phosphoramidite 32a is prepared from compound 29a according to the procedures described in Examples 5, 6 and 8. 325 WO 2004/094345 PCT/US2004/011822 Example 206 Solid support 33a (Scheme 6): The solid support 33a is prepared from compound 31a as reported in Example 10. Compound 31a is obtained from compound 29a as reported in Examples 5 and 6. 5 Example 207 Compound 29b (Scheme 6): Compound 3 is coupled to Me-O-PEGCH 2 CH2COOH in the presence of DCC and DMAP in DMF. The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29b. Example 208 10 Compound 32b (Z = Me, Scheme 6): The phosphoramidite 32b is prepared from compound 29b according to the procedures described in Examples 5, 6 and 7. Example 209 Compound 32b (Z = allyl, Scheme 6): The phosphoramidite 32b is prepared from compound 29b according to the procedures described in Examples 5, 6 and 8. 15 Example 210 Compound 32b (Z = P-cyanoethyl, Scheme 6): The phosphoramidite 32b is prepared from compound 29b according to the procedures described in Examples 5, 6 and 8. Example 211 Solid support 33b (Scheme 6): The solid support 33b is prepared from compound 31b 20 as reported in Example 10. Compound 31b is obtained from compound 29b as reported in Examples 5 and 6. Example 212 Compound 29d (n = 13, Scheme 6): Compound 3 is coupled to palmitic acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off 25 from the reaction mixture followed by standard workup to obtain compound 29d. Example 213 Compound 32d (Z = Me, n = 13, Scheme 6): The phosphoramidite 32d is prepared from compound 29d according to the procedures described in Examples 5, 6 and 7. Example 214 30 Compound 32d (Z = allyl, n = 13, Scheme 6): The phosphoramidite 32d is prepared from compound 29d according to the procedures described in Examples 5, 6 and 8. 326 WO 2004/094345 PCT/US2004/011822 Example 215 Compound 32d (Z = P-cyanoethyl, n = 13, Scheme 6): The phosphoramidite 32d is prepared from compound 29d according to the procedures described in Examples 5, 6 and 8. Example 216 5 Solid support 33d (Scheme 6): The solid support 33d is prepared from compound 31d as reported in Example 10. Compound 31d is obtained from compound 29d as reported in Examples 5 and 6. Example 217 Compound 29e (n = 6, Scheme 6): Compound 3 is coupled to N-(Fmoc)-8 10 aminooctanoic acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29e. Example 218 Compound 32e (Z = Me, n = 6, Scheme 6): The phosphoramidite 32e is prepared from 15 compound 29e according to the procedures described in Examples 5, 6 and 7. Example 219 Compound 32e (Z = allyl, n = 13, Scheme 6): The phosphoramidite 32e is prepared from compound 29e according to the procedures described in Examples 5, 6 and 8. Example 220 20 Compound 32e (Z = P-cyanoethyl, n = 6, Scheme 6): The phosphoramidite 32e is prepared from compound 29e according to the procedures described in Examples 5, 6 and 8. Example 221 Solid support 33e (Scheme 6): The solid support 33e is prepared from compound 31e as reported in Example 10. Compound 31e is obtained from compound 29e as reported in Examples 25 5 and 6. Example 222 Compound 29g (Scheme 6): Compound 3 is coupled to 0 3 -(acetyl)lithocholic acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29g. 30 Example 223 Compound 32g (Z = Me, Scheme 6): The phosphoramidite 32g is prepared from compound 29g according to the procedures described in Examples 5, 6 and 7. 327 WO 2004/094345 PCT/US2004/011822 Example 224 Compound 32g (Z = allyl, Scheme 6): The phosphoramidite 32g is prepared from compound 29g according to the procedures described in Examples 5, 6 and 8. Example 225 5 Compound 32g (Z = 0-cyanoethyl, Scheme 6): The phosphoramidite 32g is prepared from compound 29g according to the procedures described in Examples 5, 6 and 8. Example 226 Solid support 33g (Scheme 6): The solid support 33g is prepared from compound 31g as reported in Example 10. Compound 31g is obtained from compound 29g as reported in 10 Examples 5 and 6. Example 227 Compound 29h (Scheme 6): Compound 3 is coupled to pyrene butyric acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29g. 15 Example 228 Compound 32h (Z = Me, Scheme 6): The phosphoramidite 32h is prepared from compound 29h according to the procedures described in Examples 5, 6 and 7. Example 229 Compound 32h (Z = allyl, Scheme 6): The phosphoramidite 32h is prepared from 20 compound 29h according to the procedures described in Examples 5, 6 and 8. Example 230 Compound 32h (Z = P-cyanoethyl, Scheme 6): The phosphoramidite 32h is prepared from compound 29h according to the procedures described in Examples 5, 6 and 8. Example 231 25 Solid support 33h (Scheme 6): The solid support 33h is prepared from compound 31h as reported in Example 10. Compound 31h is obtained from compound 29h as reported in Examples 5 and 6. Example 232 Compound 29i (Scheme 6): Compound 3 is coupled to a,o-NFmoc-L-Lys-OH in the 30 presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29i. Example 233 Compound 32i (Z = Me, Scheme 6): The phosphoramidite 32i is prepared from compound 29i according to the procedures described in Examples 5, 6 and 7. 328 WO 2004/094345 PCT/US2004/011822 Example 234 Compound 32i (Z= allyl, Scheme 6): The phosphoramidite 32i is prepared from compound 29i according to the procedures described in Examples 5, 6 and 8. Example 235 5 Compound 32i (Z = f-cyanoethyl, Scheme 6): The phosphoramidite 32i is prepared from compound 29i according to the procedures described in Examples 5, 6 and 8. Example 236 Solid support 33i (Scheme 6): The solid support 33i is prepared from compound 31i as reported in Example 10. Compound 31i is obtained from compound 29i as reported in Examples 10 5 and6. Example 237 Compound 29j (Scheme 6): Compound 3 is coupled to a,o-NFmoc-D-Lys-OH in the presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29j. 15 Example 238 Compound 32j (Z = Me, Scheme 6): The phosphoramidite 32j is prepared from compound 29i according to the procedures described in Examples 5, 6 and 7. Example 239 Compound 32j (Z= allyl, Scheme 6): The phosphoramidite 32j is prepared from 20 compound 29j according to the procedures described in Examples 5, 6 and 8. Example 240 Compound 32j (Z = 0-cyanoethyl, Scheme 6): The phosphoramidite 32j is prepared from compound 29j according to the procedures described in Examples 5, 6 and 8. Example 241 25 Solid support 33j (Scheme 6): The solid support 33j is prepared from compound 31j as reported in Example 10. Compound 31i is obtained from compound 29j as reported in Examples 5 and 6. Example 242 Compound 291 (Scheme 6): Compound 3 is coupled to adamantane acetic acid in the 30 presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 29j. Example 243 Compound 321 (Z= Me, Scheme 6): The phosphoramidite 321 is prepared from compound 291 according to the procedures described in Examples 5, 6 and 7. 329 WO 2004/094345 PCT/US2004/011822 Example 244 Compound 321 (Z = allyl, Scheme 6): The phosphoramidite 321 is prepared from compound 291 according to the procedures described in Examples 5, 6 and 8. Example 245 5 Compound 321 (Z = P-cyanoethyl, Scheme 6): The phosphoramidite 321 is prepared from compound 291 according to the procedures described in Examples 5, 6 and 8. Example 246 Solid support 331 (Scheme 6): The solid support 331 is prepared from compound 311 as reported in Example 10. Compound 311 is obtained from compound 291 as reported in Examples 10 5 and 6. Example 247 Compound 34a (Scheme 7): Compound 6 is coupled to 0 3 -(acetyl)cholenic acid in the presence of DCC and DMAP in DMF. The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34a. 15 Example 248 Compound 37a (Z= Me, Scheme 7): The phosphoramidite 37a is prepared from compound 34a according to the procedures described in Examples 5, 6 and 7. Example 249 Compound 37a (Z = allyl, Scheme 7): The phosphoramidite 37a is prepared from 20 compound 34a according to the procedures described in Examples 5, 6 and 8. Example 250 Compound 37a (Z = 3-cyanoethyl, Scheme 7): The phosphoramidite 37a is prepared from compound 34a according to the procedures described in Examples 5, 6 and 8. Example 251 25 Solid support 38a (Scheme 7): The solid support 38a is prepared from compound 36a as reported in Example 10. Compound 36a is obtained from compound 34a as reported in Examples 5 and 6. Example 252 Compound 34b (Scheme 7): Compound 6 is coupled to Me-O-PEGCH 2

CH

2 COOH in 30 the presence of DCC and DMAP in DMF. The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34b. Example 253 Compound 37b (Z = Me, Scheme 7): The phosphoramidite 37b is prepared from compound 34b according to the procedures described in Examples 5, 6 and 7. 330 WO 2004/094345 PCT/US2004/011822 Example 254 Compound 37b (Z= allyl, Scheme 7): The phosphoramidite 37b is prepared from compound 34b according to the procedures described in Examples 5, 6 and 8. Example 255 5 Compound 37b (Z = 3-cyanoethyl, Scheme 7): The phosphoramidite 37b is prepared from compound 34b according to the procedures described in Examples 5, 6 and 8. Example 256 Solid support 38b (Scheme 7): The solid support 38b is prepared from compound 36b as reported in Example 10. Compound 36b is obtained from compound 34b as reported in 10 Examples 5 and 6. Example 257 Compound 34d (n = 13, Scheme 7): Compound 6 is coupled to palmitic acid in the presence of DCC and DMAP in DMF-CH2C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34d. 15 Example 258 Compound 37d (Z = Me, n = 13, Scheme 7): The phosphoramidite 37d is prepared from compound 34d according to the procedures described in Examples 5, 6 and 7. Example 259 Compound 37d (Z = allyl, n = 13, Scheme 7): The phosphoramidite 32d is prepared 20 from compound 34d according to the procedures described in Examples 5, 6 and 8. Example 260 Compound 37d (Z = 3-cyanoethyl, u = 13, Scheme 7): The phosphoramidite 37d is prepared from compound 34d according to the procedures described in Examples 5, 6 and 8. Example 261 25 Solid support 38d (Scheme 7): The solid support 33d is prepared from compound 36d as reported in Example 10. Compound 36d is obtained from compound 34d as reported in Examples 5 and 6. Example 262 Compound 34e (n = 6, Scheme 7): Compound 6 is coupled to N-(Fmoc)-8 30 aminooctanoic acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34e. 331 WO 2004/094345 PCT/US2004/011822 Example 263 Compound 37e (Z = Me, n = 6, Scheme 7): The phosphoramidite 37e is prepared from compound 34e according to the procedures described in Examples 5, 6 and 7. Example 264 5 Compound 37e (Z = allyl, n= 13, Scheme 7): The phosphoramidite 37e is prepared from compound 34e according to the procedures described in Examples 5, 6 and 8. Example 265 Compound 37e (Z= P3-cyanoethyl, n = 6, Scheme 7): The phosphoramidite 37e is prepared from compound 34e according to the procedures described in Examples 5, 6 and 8. 10 Example 266 Solid support 38e (Scheme 7): The solid support 37e is prepared from compound 36e as reported in Example 10. Compound 36e is obtained from compound 34e as reported in Examples 5 and 6. Example 267 15 Compound 34g (Scheme 7): Compound 6 is coupled to 0 3 -(acetyl)lithocholic acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34g. Example 268 Compound 37g (Z = Me, Scheme 7): The phosphoramidite 37g is prepared from 20 compound 34g according to the procedures described in Examples 5, 6 and 7. Example 269 Compound 37g (Z = allyl, Scheme 7): The phosphoramidite 37g is prepared from compound 3 4g according to the procedures described in Examples 5, 6 and 8. Example 270 25 Compound 37g (Z = 3-cyanoethyl, Scheme 7): The phosphoramidite 37g is prepared from compound 34g according to the procedures described in Examples 5, 6 and 8. Example 271 Solid support 38g (Scheme 7): The solid support 38g is prepared from compound 36g as reported in Example 10. Compound 36g is obtained from compound 34g as reported in 30 Examples 5 and 6. Example 272 Compound 34h (Scheme 7): Compound 6 is coupled to pyrene butyric acid in the presence of DCC and DMAP in DMF-CH 2 C1 2 (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34g. 332 WO 2004/094345 PCT/US2004/011822 Example 273 Compound 37h (Z = Me, Scheme 7): The phosphoramidite 37h is prepared from compound 34h according to the procedures described in Examples 5, 6 and 7. Example 274 5 Compound 37h (Z = allyl, Scheme 7): The phosphoramidite 37h is prepared from compound 34h according to the procedures described in Examples 5, 6 and 8. Example 275 Compound 37h (Z = (-cyanoethyl, Scheme 7): The phosphoramidite 37h is prepared from compound 34h according to the procedures described in Examples 5, 6 and 8. 10 Example 276 Solid support 38h (Scheme 7): The solid support 38h is prepared from compound 36h as reported in Example 10. Compound 36h is obtained from compound 34h as reported in Examples 5 and 6. Example 277 15 Compound 34i (Scheme 7): Compound 6 is coupled to a,co-NFmoc-L-Lys-OH in the presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34i. Example 278 Compound 37i (Z = Me, Scheme 7): The phosphoramidite 37i is prepared from 20 compound 34i according to the procedures described in Examples 5, 6 and 7. Example 279 Compound 37i (Z= allyl, Scheme 7): The phosphoramidite 37i is prepared from compound 34i according to the procedures described in Examples 5, 6 and 8. Example 280 25 Compound 37i (Z = P3-cyanoethyl, Scheme 7): The phosphoramidite 37i is prepared from compound 34i according to the procedures described in Examples 5, 6 and 8. Example 281 Solid support 38i (Scheme 7): The solid support 38i is prepared from compound 36i as reported in Example 10. Compound 36i is obtained from compound 34i as reported in Examples 30 5 and 6. Example 282 Compound 34j (Scheme 7): Compound 6 is coupled to a.,o-NFmc-D-Lys-OH in the presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 34j. 333 WO 2004/094345 PCT/US2004/011822 Example 283 Compound 34j (Z= Me, Scheme 7): The phosphoramidite 37j is prepared from compound 34i according to the procedures described in Examples 5, 6 and 7. Example 284 5 Compound 37j (Z = allyl, Scheme 7): The phosphoramidite 37j is prepared from compound 34j according to the procedures described in Examples 5, 6 and 8. Example 285 Compound 37j (Z = 0-cyanoethyl, Scheme 7): The phosphoramidite 37j is prepared from compound 34j according to the procedures described in Examples 5, 6 and 8. 10 Example 286 Solid support 38j (Scheme 7): The solid support 38j is prepared from compound 36j as reported in Example 10. Compound 36i is obtained from compound 34j as reported in Examples 5 and 6. Example 287 15 Compound 341 (Scheme 7): Compound 6 is coupled to adamantane acetic acid in the presence of HATU and HOAT in DMF (1:1 mixture). The precipitated DCU is filtered off from the reaction mixture followed by standard workup to obtain compound 341. Example 288 Compound 371 (Z = Me, Scheme 7): The phosphoramidite 371 is prepared from 20 compound 341 according to the procedures described in Examples 5, 6 and 7. Example 289 Compound 371 (Z = allyl, Scheme 7): The phosphoramidite 371 is prepared from compound 341 according to the procedures described in Examples 5, 6 and 8. Example 290 25 Compound 371 (Z = 0-cyanoethyl, Scheme 7): The phosphoramidite 371 is prepared from compound 341 according to the procedures described in Examples 5, 6 and 8. Example 291 Solid support 381 (Scheme 7): The solid support 381 is prepared from compound 361 as reported in Example 10. Compound 361 is obtained from compound 341 as reported in Examples 30 5 and 6. Example 292 Compound 39a (n = 14, Scheme 8): Hexadecylamine (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 6 (1 mmol) is added into the reaction 334 WO 2004/094345 PCT/US2004/011822 mixture after 2 h and the stirring is continued for overnight to obtain compound 39a (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). Example 293 Compound 42a (n = 14,Z = Me, Scheme 8): The phosphoramidite 42a is prepared from 5 compound 39a according to the procedures described in Examples 5, 6 and 7. Example 294 Compound 42a (n = 14, Z = allyl, Scheme 8): The phosphoramidite 42a is prepared from compound 39a according to the procedures described in Examples 5, 6 and 8. Example 295 10 Compound 42a (n = 14, Z = 1-cyanoethyl, Scheme 8): The phosphoramidite 42a is prepared from compound 39a according to the procedures described in Examples 5, 6 and 8. Example 296 Solid support 43a (n = 14, Scheme 8): The solid support 43a is prepared from compound 41a as reported in Example 10. Compound 41a is obtained from compound 39a as 15 reported in Examples 5 and 6. Example 297 Compound 39b (n = 7, Scheme 8): N1-(Fmoc)-1,8-octanediamine (1 rmmol) in anhydrous THF is stirred with 1,1 '-carbonyldiirnidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 6 (1 mmol) is added 20 into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 39b (Hemandez and Hodges, J Org. Chem., 1997, 62, 3153-3157). Example 298 Compound 42b (Z = Me, n = 7, Scheme 8): The phosphoramidite 42b is prepared from compound 39b according to the procedures described in Examples 5, 6 and 7. 25 Example 299 Compound 42b (Z = allyl, n = 7, Scheme 8): The phosphoramidite 42b is prepared from compound 39b according to the procedures described in Examples 5, 6 and 8. Example 300 Compound 42b (Z = P-cyanoethyl, n = 7, Scheme 8): The phosphoramidite 42b is 30 prepared from compound 39b according to the procedures described in Examples 5, 6 and 8. Example 301 Solid support 43b (n = 7, Scheme 8): The solid support 43b is prepared from compound 41b as reported in Example 10. Compound 41b is obtained from compound 39b as reported in Examples 5 and 6. 335 WO 2004/094345 PCT/US2004/011822 Example 302 Compound 39c (n = 3, Scheme 8): N -(COCF 3 )-1,8-octanediamine (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 6 (1 mmol) is added 5 into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 39c (Hernandez and Hodges, J Org. Chem., 1997, 62, 3153-3157). Example 303 Compound 42c (Z = Me, n= 3, Scheme 8): The phosphoramidite 42c is prepared from compound 39c according to the procedures described in Examples 5, 6 and 7. 10 Example 304 Compound 42c (Z = allyl, n = 3, Scheme 8): The phosphoramidite 42c is prepared from compound 39c according to the procedures described in Examples 5, 6 and 8. Example 305 Compound 42c (Z = 3-cyanoethyl, n = 3, Scheme 8): The phosphoramidite 42c is 15 prepared from compound 39c according to the procedures described in Examples 5, 6 and 8. Example 306 Solid support 43c (n = 3, Scheme 8): The solid support 43c is prepared from compound 41c as reported in Example 10. Compound 41c is obtained from compound 39c as reported in Examples 5 and 6. 20 Example 307 Compound 39d (n = 3, Scheme 8): 0 4 -(Acetyl)-4-hydroxy-1-aminobutane (1 mmol) in anhydrous THF is stirred with 1,1 '-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 6 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 25 39d (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). Example 308 Compound 42d (Z = Me, n = 3, Scheme 8): The phosphoramidite 42d is prepared from compound 39d according to the procedures described in Examples 5, 6 and 7. Example 309 30 Compound 42d (Z = allyl, n = 3, Scheme 8): The phosphoramidite 42d is prepared from compound 39d according to the procedures described in Examples 5, 6 and 8. 336 WO 2004/094345 PCT/US2004/011822 Example 310 Compound 42d (Z = -cyanoethyl, n = 3, Scheme 8): The phosphoramidite 42d is prepared from compound 39d according to the procedures described in Examples 5, 6 and 8. 5 Example 311 Solid support 43d (n = 3, Scheme 8): The solid support 43d is prepared from compound 41d as reported in Example 10. Compound 41d is obtained from compound 39d as reported in Examples 5 and 6. Example 312 10 Compound 39e (n = 2, Scheme 8): y-Aminobutyric acid ethyl ester (1 mmol) is stirred with CDI (1 mmol) in THF at ambient temperature for overnight. After overnight stirring, compound 6 is added into the reaction mixture and continued stirring for overnight to obtain compound 39e. Example 313 15 Compound 42e (Z = Me, n = 2, Scheme 8): The phosphoramidite 42e is prepared from compound 39e according to the procedures described in Examples 5, 6 and 7. Example 314 Compound 42e (Z = allyl, n = 2, Scheme 8): The phosphoramidite 42e is prepared from compound 39e according to the procedures described in Examples 5, 6 and 8. 20 Example 315 Compound 42e (Z = P-cyanoethyl, n = 2, Scheme 8): The phosphoramidite 42e is prepared from compound 39e according to the procedures described in Examples 5, 6 and 8. Example 316 Solid support 43e (n = 2, Scheme 8): The solid support 43e is prepared from compound 25 41e as reported in Example 10. Compound 41e is obtained from compound 39e as reported in Examples 5 and 6. Example 317 Compound 39g (n = 2, Scheme 8): N 3 -(CONH2)-l,3-diaminopropane (1 mmol) in anhydrous THF is stirred with 1,1 '-carbonyldiimidazole (CDI, 1 mmol) in the presence of 30 DMAP at ambient temperature under argon atmosphere for 2 h. Compound 6 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 39g (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). 337 WO 2004/094345 PCT/US2004/011822 Example 318 Compound 42g (Z = Me, n = 2, Scheme 8): The phosphoramidite 4 2 g is prepared from compound 39g according to the procedures described in Examples 5, 6 and 7. Example 319 5 Compound 42g (Z = allyl, n = 2, Scheme 8): The phosphoramidite 42g is prepared from compound 39g according to the procedures described in Examples 5, 6 and 8. Example 320 Compound 42g (Z= 03-cyanoethyl, n= 2, Scheme 8): The phosphoramidite 42g is prepared from compound 39g according to the procedures described in Examples 5, 6 and 8. 10 Example 321 Solid support 43g (n = 2, Scheme 8): The solid support 43g is prepared from compound 41g as reported in Example 10. Compound 41g is obtained from compound 39g as reported in Examples 5 and 6. Example 322 15 Compound 44a (n = 14, Scheme 9): Hexadecylamine (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 44a (Hernandez and Hodges, J Org. Chem., 1997, 62, 3153-3157). 20 Example 323 Compound 47a (n = 14,Z= Me, Scheme 9): The phosphoramidite 47a is prepared from compound 44a according to the procedures described in Examples 5, 6 and 7. Example 324 Compound 47a (n = 14, Z = allyl, Scheme 9): The phosphoramidite 49a is prepared 25 from compound 44a according to the procedures described in Examples 5, 6 and 8. Example 325 Compound 47a (n = 14, Z = P3-cyanoethyl, Scheme 9): The phosphoramidite 47a is prepared from compound 44a according to the procedures described in Examples 5, 6 and 8. Example 326 30 Solid support 48a (n = 14, Scheme 9): The solid support 48a is prepared from compound 46a as reported in Example 10. Compound 46a is obtained from compound 44a as reported in Examples 5 and 6. 338 WO 2004/094345 PCT/US2004/011822 Example 327 Compound 44b (n = 7, Scheme 9): N-(Fmoc)-1,8-octanediamine (1 rmmol) in anhydrous THF is stirred with 1,1 '-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added 5 into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 44b (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). Example 328 Compound 47b (Z= Me, n = 7, Scheme 9): The phosphoramidite47b is prepared from compound 44b according to the procedures described in Examples 5, 6 and 7. 10 Example 329 Compound 47b (Z= allyl, n = 7, Scheme 9): The phosphoramidite 47b is prepared from compound 44b according to the procedures described in Examples 5, 6 and 8. Example 330 Compound 47b (Z= (3-cyanoethyl, n = 7, Scheme 9): The phosphoramidite 47b is 15 prepared from compound 44b according to the procedures described in Examples 5, 6 and 8. Example 331 Solid support 48b (n = 7, Scheme 9): The solid support 48b is prepared from compound 46b as reported in Example 10. Compound 46b is obtained from compound 44b as reported in Examples 5 and 6. 20 Example 332 Compound 44c (n = 3, Scheme 9): N-(COCF 3 )-1,8-octanediamine (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 25 44c (Hernandez and Hodges, J Org. Chem., 1997, 62, 3153-3157). Example 333 Compound 47c (Z = Me, n = 3, Scheme 9): The phosphoramidite 49c is prepared from compound 44c according to the procedures described in Examples 5, 6 and 7. Example 334 30 Compound 47c (Z = allyl, n = 3, Scheme 9): The phosphoramidite 47c is prepared from compound 44c according to the procedures described in Examples 5, 6 and 8. 339 WO 2004/094345 PCT/US2004/011822 Example 335 Compound 47c (Z = -cyanoethyl, n = 3, Scheme 9): The phosphoramidite 47c is prepared from compound 44c according to the procedures described in Examples 5, 6 and 8. Example 336 5 Solid support 48c (n = 3, Scheme 9): The solid support 48c is prepared from compound 46c as reported in Example 10. Compound 46c is obtained from compound 44c as reported in Examples 5 and 6. Example 337 Compound 44d (n = 3, Scheme 9): 0 4 -(Acetyl)-4-hydroxy-1-aminobutane (1 mmol) in 10 anhydrous THF is stirred with 1, l'-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 44d (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). Example 338 15 Compound 47d (Z = Me, n = 3, Scheme 9): The phosphoramidite 47d is prepared from compound 44d according to the procedures described in Examples 5, 6 and 7. Example 339 Compound 47d (Z= allyl, n = 3, Scheme 9): The phosphoramidite 47d is prepared from compound 44d according to the procedures described in Examples 5, 6 and 8. 20 Example 340 Compound 47d (Z = i-cyanoethyl, n = 3, Scheme 9): The phosphoramidite 47d is prepared from compound 44d according to the procedures described in Examples 5, 6 and 8. Example 341 Solid support 48d (n = 3, Scheme 9): The solid support 48d is prepared from compound 25 46d as reported in Example 10. Compound 46d is obtained from compound 44d as reported in Examples 5 and 6. Example 342 Compound 44e (n = 2, Scheme 9): y-Aminobutyric acid ethyl ester (1 mmol) in anhydrous THF is stirred with 1,1'-carbonyldiimidazole (CDI, 1 mmol) in the presence of 30 DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 44e (Hernandez and Hodges, J. Org. Chemn., 1997, 62, 3153-3157). 340 WO 2004/094345 PCT/US2004/011822 Example 343 Compound 47e (Z = Me, n = 2, Scheme 9): The phosphoramidite 47e is prepared from compound 44e according to the procedures described in Examples 5, 6 and 7. Example 344 5 Compound 47e (Z = allyl, n = 2, Scheme 9): The phosphoramidite 47e is prepared from compound 44e according to the procedures described in Examples 5, 6 and 8. Example 345 Compound 47e (Z = P-cyanoethyl, n = 2, Scheme 9): The phosphoramidite 47e is prepared from compound 44e according to the procedures described in Examples 5, 6 and 8. 10 Example 346 Solid support 48e (n = 2, Scheme 9): The solid support 48e is prepared from compound 46e as reported in Example 10. Compound 46e is obtained from compound 44e as reported in Examples 5 and 6. Example 347 15 Compound 44g (n = 2, Scheme 9): N 3 -(CONH2)-1,3-diaminopropane (1 mmol) in anhydrous THF is stirred with 1,1 '-carbonyldiimidazole (CDI, 1 mmol) in the presence of DMAP at ambient temperature under argon atmosphere for 2 h. Compound 3 (1 mmol) is added into the reaction mixture after 2 h and the stirring is continued for overnight to obtain compound 44g (Hernandez and Hodges, J. Org. Chem., 1997, 62, 3153-3157). 20 Example 348 Compound 4 7g (Z= Me, n = 2, Scheme 9): The phosphoramidite 4 7g is prepared from compound 44g according to the procedures described in Examples 5, 6 and 7. Example 349 Compound 47g (Z= allyl, n = 2, Scheme 9): The phosphoramidite 4 7g is prepared from 25 compound 44g according to the procedures described in Examples 5, 6 and 8. Example 350 Compound 47g (Z = 3 -cyanoethyl, n = 2, Scheme 9): The phosphoramidite 47g is prepared from compound 44g according to the procedures described in Examples 5, 6 and 8. Example 351 30 Solid support 48g (n = 2, Scheme 9): The solid support 4 8g is prepared from compound 46g as reported in Example 10. Compound 46g is obtained from compound 44g as reported in Examples 5 and 6. 341 WO 2004/094345 PCT/US2004/011822 Example 352 Compound 49a-1 (Scheme 10): Compound 8a-I (Scheme 2, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 49a-1 respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). 5 Example 353 Compound 50a-I (Z = Me, Scheme 10): The phosphoramidites 50a-I are prepared from compounds 49a-1 respectively as reported in Example 7. Example 354 Compound 50a-I (Z= allyl, Scheme 10): The phosphoramidites 50a-I are prepared from 10 compounds 49a-1 respectively as reported in Example 8. Example 355 Compound 50a-I (Z = 03-cyanoethyl, Scheme 10): The phosphoramidites 50a-I are prepared from compounds 49a-I respectively as reported in Example 9. Example 356 15 Solid support 51a-l (Scheme 10): The solid supports 51a-I are prepared from compounds 49a-1 respectively as reported in Example 10. Example 357 Compound 52a-I (Scheme 11): Compound 13a-1 (Scheme 3, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives 20 compound 52a-1 respectively (Rouch and Russo-Rodriguez, J. Org. Chem., 1987, 52, 598). Example 358 Compound 53a-1 (Z= Me, Scheme 11): The phosphoramidites 53a-1 are prepared from compounds 52a-1 respectively as reported in Example 7. Example 359 25 Compound 53a-1 (Z= allyl, Scheme 11): The phosphoramidites 53a-1 are prepared from compounds 52a-1 respectively as reported in Example 8. Example 360 Compound 53a-1 (Z= 3-cyanoethyl, Scheme 11): The phosphoramidites 53a-1 are prepared from compounds 52a-1 respectively as reported in Example 9. 30 Example 361 Solid support 54a-I (Scheme 11): The solid supports 54a-1 are prepared from compounds 52a-1 respectively as reported in Example 10. 342 WO 2004/094345 PCT/US2004/011822 Example 362 Compound 55a-1 (Scheme 12): Compound 19a-1 (Scheme 4, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 55a-1 respectively (Rouch and Russo-Rodriguez, J. Org. Chem., 1987, 52, 598). 5 Example 363 Compound 56a-I (Z = Me, Scheme 12): The phosphoramidites 56a-I are prepared from compounds 55a-1 respectively as reported in Example 7. Example 364 Compound 56a-1 (Z = allyl, Scheme 12): The phosphoramidites 56a-1 are prepared from o10 compounds 55a-1 respectively as reported in Example 8. Example 365 Compound 56a-1 (Z = j-cyanoethyl, Scheme 12): The phosphoramidites 56a-1 are prepared from compounds 55a-I respectively as reported in Example 9. Example 366 15 Solid support 57a-I (Scheme 12): The solid supports 57a-1 are prepared from compounds 55a-I respectively as reported in Example 10. Example 367 Compound 58a-1 (Scheme 13): Compound 25a-1 (Scheme 5, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives 20 compound 58a-l respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). Example 368 Compound 59a-1 (Z = Me, Scheme 13): The phosphoramidites 59a-1 are prepared from compounds 58a-l respectively as reported in Example 7. Example 369 25 Compound 59a-1 (Z= allyl, Scheme 13): The phosphoramidites 59a-1 are prepared from compounds 58a-l respectively as reported in Example 8. Example 370 Compound 59a-1 (Z = 3-cyanoethyl, Scheme 13): The phosphoramidites 59a-I are prepared from compounds 58a-l respectively as reported in Example 9. 30 Example 371 Solid support 60a-I (Scheme 13): The solid supports 60a-l are prepared from compounds 58a-1 respectively as reported in Example 10. 343 WO 2004/094345 PCT/US2004/011822 Example 367 Compound 5 8a-1 (Scheme 13): Compound 25a-1 (Scheme 5, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 58a-1 respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). 5 Example 368 Compound 59a-1 (Z = Me, Scheme 13): The phosphoramidites 59a-1 are prepared from compounds 58a-l respectively as reported in Example 7. Example 369 Compound 59a-I (Z= allyl, Scheme 13): The phosphoramidites 59a-1 are prepared from 10 compounds 58a-l respectively as reported in Example 8. Example 370 Compound 59a-1 (Z = 0-cyanoethyl, Scheme 13): The phosphoramidites 59a-I are prepared from compounds 58a-I respectively as reported in Example 9. Example 371 15 Solid support 60a-I (Scheme 13): The solid supports 60a-I are prepared from compounds 58a-1 respectively as reported in Example 10. Example 372 Compound 61a-1 (Scheme 14): Compound 30a-I (Scheme 6, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives 20 compound 61a-l respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). Example 373 Compound 62a-1 (Z = Me, Scheme 14): The phosphoramidites 62a-1 are prepared from compounds 61a-I respectively as reported in Example 7. Example 374 25 Compound 62a-1 (Z = allyl, Scheme 14): The phosphoramidites 62a-1 are prepared from compounds 61a-I respectively as reported in Example 8. Example 375 Compound 62a-1 (Z= 1-cyanoethyl, Scheme 14): The phosphoramidites 62a-1 are prepared from compounds 61a-I respectively as reported in Example 9. 30 Example 376 Solid support 60a-I (Scheme 14): The solid supports 63a-1 are prepared from compounds 61a-1 respectively as reported in Example 10. Example 377 344 WO 2004/094345 PCT/US2004/011822 Compound 64a-1 (Scheme 15): Compound 30a-1 (Scheme 7, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 64a-1 respectively (Rouch and Russo-Rodriguez, J. Org. Chem., 1987, 52, 598). Example 378 5 Compound 65a-1 (Z = Me, Scheme 15): The phosphoramidites 65a-1 are prepared from compounds 64a-1 respectively as reported in Example 7. Example 379 Compound 65a-1 (Z = allyl, Scheme 15): The phosphoramidites 65a-1 are prepared from compounds 64a-1 respectively as reported in Example 8. 10 Example 380 Compound 65a-1 (Z = P-cyanoethyl, Scheme 15): The phosphoramidites 65a-1 are prepared from compounds 64a-I respectively as reported in Example 9. Example 381 Solid support 66a-1 (Scheme 15): The solid supports 66a-I are prepared from 15 compounds 64a-1 respectively as reported in Example 10. Example 382 Compound 67a-g (Scheme 16): Compound 40a-g (Scheme 8, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 67a-g respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). 20 Example 383 Compound 68a-g (Z= Me, Scheme 16): The phosphoramidites 68a-g are prepared from compounds 67a-g respectively as reported in Example 7. Example 384 Compound 68a-g (Z = allyl, Scheme 16): The phosphoramidites 68a-g are prepared 25 from compounds 67a-g respectively as reported in Example 8. Example 385 Compound 68a-g (Z= O-cyanoethyl, Scheme 16): The phosphoramidites 68a-g are prepared from compounds 67a-g respectively as reported in Example 9. Example 386 30 Solid support 69a-g (Scheme 16): The solid supports 69a-g are prepared from compounds 67a-g respectively as reported in Example 10. 345 WO 2004/094345 PCT/US2004/011822 Example 387 Compound 70a-g (Scheme 17): Compound 45a-g (Scheme 9, Examples) in anhydrous THF is stirred respectively with triethylsilyl chloride (TES-C1) in the presence of imidazole gives compound 70a-g respectively (Rouch and Russo-Rodriguez, J Org. Chem., 1987, 52, 598). 5 Example 388 Compound 71a-g (Z= Me, Scheme 17): The phosphoramidites 71a-g are prepared from compounds 70a-g respectively as reported in Example 7. Example 389 Compound 71a-g (Z= allyl, Scheme 17): The phosphoramidites 71a-g are prepared 10 from compounds 70a-g respectively as reported in Example 8. Example 390 Compound 71a-g (Z= j3-cyanoethyl, Scheme 17): The phosphoramidites 71a-g are prepared from compounds 70a-g respectively as reported in Example 9. Example 391 15 Solid support 72a-g (Scheme 17): The solid supports 72a-g are prepared from compounds 70a-g respectively as reported in Example 10. Example 392 Compounds 1002a-r (Scheme 1001): Nucleosides 1001a-s are obtained according to literature reports (Limbach, et al., Nucleic Acids Res., 1994, 22, 2183-96). Exocyclic amino, 20 hydroxyl and/or amidino group(s) of the heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. Base protected nucleoside Is is obtained according to literature procedure (Zhao and Baranger, J Am. Chem. Soc., 2002). Suitably base protected nucleosides 1 is treated with 1,3 dichloro-1,1,3,3-tetraisopropyldisiloxane as reported in the literature to obtain the corresponding 25 3',5'-di-O-cyclic silyl derivative (Evans et al., Tetrahedron, 2000, 56, 3053-62). The desired compound 1002 is obtained from the cyclic silyl compound according to literature procedures (Chui et al., Bioorg. Med. Chem., 2002, 10, 325-332). Example 393 Compound 1003a-s (X = Me, Scheme 1001): Treatment of compounds 1002a-s with 30 methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH2C1 2 yields the corresponding phosphoramidite 1003a-s (Chui et al. J. Org. Chem., 2002, 67, 8847-54). 346 WO 2004/094345 PCT/US2004/011822 Example 394 Compound 1003a-s (X = allyl, Scheme 1001): To a solution of compound 1002 (1 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution 5 is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1003 (Hayakawa et al., J Am. Chem. Soc., 1990, 112, 1691-96). Example 395 Compound 1003a-s (X = P-cyanoethyl, Scheme 1001): The desired phosphoramidite 1003 is prepared from the corresponding precursor 1002 and -cyanoethyl tetraisopropyl 10 phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 396 Solid supports 1004a-s (R', R" = OSiMe 3 , R"' = OCH(C 6 Hs) 2 , Scheme 1001): Compound 1002 (1 mmol) is mixed with succinic anhydride (2 mmol) and 15 dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethne (0.9 mL) is added into the mixture, and the mixture is stirred at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid 20 derivative. The succinic acid derivative (1 mmol) thus obtained is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H-benzotriazole-1-yl)-1,1,3,3 tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4-methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution and allows to shake on a shaker at ambient temperature for 18 h. An 25 aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 30 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The final loading capacity of the corresponding solid support 1004 is determined after capping (Prakash et aL., J Org. Chem. 2002, 67, 357-369). 347 WO 2004/094345 PCT/US2004/011822 Example 397 Compound 1005a-s (Scheme 1001): Nucleosides 1001a-s are obtained according to literature reports (Limbach, et al., Nucleic Acids Res., 1994, 22, 2183-96). Exocyclic amino, hydroxyl and/or amidino group(s) of the heterocyclic base is(are) appropriately protected for 5 solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. Base protected nucleoside Is is obtained according to literature procedure (Zhao and Baranger, J Am. Chem. Soc., 2002). Suitably base protected nucleosides 1 is treated with 1,3 dichloro-1,l,3,3-tetraisopropyldisiloxane as reported in the literature to obtain the corresponding 3',5'-di-O-cyclic silyl derivative (Evans et al., Tetrahedron, 2000, 56, 3053-62). The cyclic silyl 10 is treated with tris(2-acetoxyethoxy)orthoformate, pyridinium p-toluenesulfonate, 4-(tert butyldimethylsilyloxy)-3-penten-2-one in dioxane as reported by Chui et al. (Biorg. Med. Chem., 2002, 10, 325-332) to obtain the corresponding 2'-O- bis(2-acetoxyethoxy)methyl protected nucleoside. The completely protected nucleoside thus obtained is treated with TMEDA-HF in

CH

3 CN (Chui et al. Biorg. Med. Chem., 2002, 10, 325-332) to remove the cyclic silyl protecting 15 group. Treatment of the diol obtained with triethylsilyl chloride in the presence of imidazole in acetonitrile yields the corresponding nucleoside 1005 (Oppolzer et al., Helv. Chim. Acta, 1981, 64, 2002). Example 398 Compound 1006a-s (X = Me, Scheme 1001): Treatment of compounds 1005a-u with 20 methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2

C

2 yield the corresponding phosphoramidite 1006a-s (Chui et al. J Org. Chem., 2002, 67, 8847-54). Example 399 Compound 1006a-s (X = allyl, Scheme 1001): To a solution of compound 1005a-s (1 25 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1006 (Hayakawa et al., J. Am. Chem. Soc., 1990, 112, 1691-96). Example 400 30 Compound 1006a-u (X = 03-cyanoethyl, Scheme 1001): The desired phosphoramidite 1006 is prepared from the corresponding precursor 1005 and P-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J Org. Chem. 2002, 67, 357-369). 348 WO 2004/094345 PCT/US2004/011822 Example 401 Solid supports 1004a-u (R', R", R"' = Et, Scheme 1001): Compound 1005 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over

P

2 0 5 in vacuo overnight. Dichloromethne (0.9 mL) is added into the mixture and stirs at ambient 5 temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H-benzotriazole-1-yl) 10 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4-methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution, and the mixture is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading capacity is determined by following standard 15 procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with

CH

3 CN followed by diethylether, and is dried in vacuo. The final loading capacity of the 20 corresponding solid support 1004 is determined after capping (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 402 Compounds 1008a-u (Scheme 1002): Nucleosides 1007a-u are obtained according to literature reports. Exocyclic amino, hydroxyl and/or amidino group(s) of the heterocyclic base 25 is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. Suitably base protected nucleosides 1008 are treated with 1, 3 -dichloro-1,1,3,3-tetraisopropyldisiloxane as reported in the literature to obtain the corresponding 3',5'-di-O-cyclic silyl derivative (Evans et al., Tetrahedron, 2000, 56, 3053-62). The desired compound 1007 is obtained from the cyclic silyl compound according to literature 30 procedures (Chui et al., Bioorg. Med. Chem., 2002, 10, 325-332). Example 403 Compound 1009a-u (X = Me, Scheme 1002): Treatment of compounds 1008a-u with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient 349 WO 2004/094345 PCT/US2004/011822 temperature in CH 2 C1 2 yield the corresponding phosphoramidite 1009a-u (Chui et al. J. Org. Chem., 2002, 67, 8847-54). Example 404 Compound 1009a-u (X = allyl, Scheme 1002): To a solution of compound 1002 (1 5 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1009 (Hayakawa et al., J. Am. Chem. Soc., 1990, 112, 1691-96). Example 405 10 Compound 1009a-s (X = P-cyanoethyl, Scheme 1002): The desired phosphoramidite 1009 is prepared from the corresponding precursor 1008 and P3-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 406 15 Solid supports 1010a-s (R', R"= OSiMe 3 , R"' = OCH(C 6 Hs) 2 , Scheme 1002): Compound 1008 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethne (0.9 mL) is added into the mixture, and the mixture is stired at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice 20 cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H-benzotriazole-l-yl)-l,1,3,3 tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4 -methylmorpholine (2 mmol) with 25 vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution and is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and is dried in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. 30 Unfunctionalized sites on the support are capped by shaking with acetic anhydride/collidine/N methylimidazole in THBF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The final loading capacity of the corresponding solid support 1010 is determined after capping (Prakash et al., J. Org. Chem. 2002, 67, 357-369). 350 WO 2004/094345 PCT/US2004/011822 Example 407 Compound 1011a-s (Scheme 1002): Nucleosides 1011a-u are obtained according to literature reports. Exocyclic amino, hydroxyl and/or amidino group(s) of the heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard 5 protection and deprotection protocols. Suitably base protected nucleosides 1007 is treated with 1, 3 -dichloro-1,1,3,3-tetraisopropyldisiloxane as reported in the literature to obtain the corresponding 3',5'-di-O-cyclic silyl derivative (Evans et al, Tetrahedron, 2000, 56, 3053-62). The cyclic silyl is treated with tris( 2 -acetoxyethoxy)orthoformate, pyridiniump toluenesulfonate, 4 -(tert-butyldimethylsilyloxy)-3-penten-2-one in dioxane as reported by Chui 10 et al. (Biorg. Med. Chem., 2002, 10, 325-332) to obtain the corresponding 2'-O- bis(2 acetoxyethoxy)methyl protected nucleoside. The completely protected nucleoside thus obtained is treated with TMEDA-HF in CH 3 CN (Chui et al. Biorg. Med. Chem., 2002, 10, 325-332) to remove the cyclic silyl protecting group. Treatment of the diol obtained with triethylsilyl chloride in the presence of imidazole in acetonitrile yields the corresponding nucleoside 1011 15 (Oppolzer et al., HeIv. Chim. Acta, 1981, 64, 2002). Example 408 Compound 1012a-s (X = Me, Scheme 1001): Treatment of compounds 1011a-u with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2

C

2 yields the corresponding phosphoramidite 1012a-s (Chui et aL J Org. 20 Chem., 2002, 67, 8847-54). Example 409 Compound 1012a-s (X = allyl, Scheme 1001): To a solution of compound 1011a-s (1 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution 25 is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1012 (Hayakawa et al., J. Am. Chem. Soc., 1990, 112, 1691-96). Example 410 Compound 1012a-u (X = 0-cyanoethyl, Scheme 1002): The desired phosphoramidite 1012 is prepared from the corresponding precursor 1011 and P-cyanoethyl tetraisopropyl 30 phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 411 Solid supports 1010a-u (R', R", R"' = Et, Scheme 1001): Compound 1011 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over 351 WO 2004/094345 PCT/US2004/011822

P

2 0 5 in vacuo overnight. Dichloromethane (0.9 mL) is added into the mixture and is stirred at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the 5 corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H benzotriazole-l-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4 methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution, and the mixture is allowed to shake 10 on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B 15 solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The final loading capacity of the corresponding solid support 1010 is determined after capping (Prakash et al., J Org. Chenm. 2002, 67, 357-369). Example 412 20 Compounds 1014a-j (Scheme 1003): Compounds 1013a-e (For a-d, Y = OH and for e, Y = H) are obtained as reported in the literature (Crimmins, M. T., Tetrahedron, 1998, 54, 9229 9272). Compounds 1013f and 1013g (Y = OH) are prepared according to literature reports (Rajappan and Schneller, Tetrahedron, 2001, 57, 9049-9053). Compounds 1013h-j (Y = H or OH) are obtained as reported in the literature (Borthwick and Biggadike, Tetrahedron, 1992, 48, 25 5 7 1-623).Exocyclic amino of the heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. Suitably base protected nucleosides 1013 is treated with 1,3-dichloro-1,l1, 3

,

3 -tetraisopropyldisiloxane as reported in the literature to obtain the corresponding 3',5'-di-O-cyclic silyl derivative (Evans et al., Tetrahedron, 2000, 56, 3053-62). The desired compound 1014 is obtained from the cyclic 30 silyl compound according to literature procedures (Chui et al., Bioorg. Med. Chemh., 2002, 10, 325-332). Example 413 Compound 1015a-j (X = Me, Scheme 1003): Treatment of compounds 1014a-j with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient 352 WO 2004/094345 PCT/US2004/011822 temperature in CH 2

C

2 yield the corresponding phosphoramidite 1015a-j (Chui et al. J. Org. Chem., 2002, 67, 8847-54). Example 414 Compound 1015a-j (X = allyl, Scheme 1003): To a solution of compound 1014 (1 5 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1014 (Hayakawa et aL., J. Am. Chem. Soc., 1990, 112, 1691-96). Example 415 10 Compound 1015a-j (X = P-cyanoethyl, Scheme 1003): The desired phosphoramidite 1015 is prepared from the corresponding precursor 1014 and 3-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 416 15 Solid supports 1016a-j (R', R"= OSiMe 3 , R"' = OCH(C 6 Hs) 2 , Scheme 1003): Compound 1014 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethane (0.9 mL) is added into the mixture and stirs at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric 20 acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous DMF, mixed with 2-(1H-benzotriazole-1-yl)-1,1, 3 ,3-tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4-methylmorpholine (2 mmol) with vortexing to give a 25 clear solution. Calculated amount of solid support with an amino tether is added into the clear solution and is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the 30 support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The final loading capacity of the corresponding solid support 1016 is determined after capping (Prakash et aL., J. Org. Chem. 2002, 67, 357-369). 353 WO 2004/094345 PCT/US2004/011822 Example 417 Compound 1017a-j (Scheme 1003): Compounds 1013a-e (For a-d, Y = OH and for e, Y = H) are obtained as reported in the literature (Crimmins, M. T., Tetrahedron, 1998, 54, 9229 9272). Compounds 1013f and 1013g (Y = OH) are prepared according to literature reports 5 (Rajappan and Schneller, Tetrahedron, 2001, 57, 9049-9053). Compounds 1013h-j (Y = H or OH) are obtained as reported in the literature (Borthwick and Biggadike, Tetrahedron, 1992, 48, 571-623).Exocyclic amino of the heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. Suitably base protected nucleosides 1013 is treated with 1, 3 -dichloro-l,l,3,3-tetraisopropyldisiloxane as 10 reported in the literature to obtain the corresponding 3',5'-di-O-cyclic silyl derivative (Evans et al., Tetrahedron, 2000, 56, 3053-62). The cyclic silyl is treated with tris(2 acetoxyethoxy)orthoformate, pyridiniump-toluenesulfonate, 4-(tert-butyldimethylsilyloxy)-3 penten-2-one in dioxane as reported by Chui et al. (Biorg. Med. Chem., 2002, 10, 325-332) to obtain the corresponding 2'-O- bis(2-acetoxyethoxy)methyl protected nucleoside. The 15 completely protected nucleoside thus obtained is treated with TMEDA-HF in CH3CN (Chui et al. Biorg. Med. Chem., 2002, 10, 325-332) to remove the cyclic silyl protecting group. Treatment of the diol obtained with triethylsilyl chloride in the presence of imidazole in acetonitrile yields the corresponding nucleoside 1017 (Oppolzer et aL., Helv. Chim. Acta, 1981, 64, 2002). Example 418 20 Compound 1018a-j (X = Me, Scheme 1003): Treatment of compounds 1017a-j with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2 C1 2 yield the corresponding phosphoramidite 1018a-s (Chui et al. J. Org. Chem., 2002, 67, 8847-54). Example 419 25 Compound 1018a-sj (X = allyl, Scheme 1003): To a solution of compound 1017a-j (1 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 rmmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1018 (Hayakawa et al., J Am. Chem. Soc., 1990, 112, 1691-96). 30 Example 420 Compound 1018a-j (X = 3-cyanoethyl, Scheme 1003): The desired phosphoramidite 1018 is prepared from the corresponding precursor 1017 and P3-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). 354 WO 2004/094345 PCT/US2004/011822 Example 421 SOLID SUPPORT supports 1016a-u (R', R", R"'= Et, Scheme 1003): Compound 1017 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethane (0.9 mL) is added into the 5 mixture and stirs at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum overnight and suspended in anhydrous 10 DMF, mixed with 2-(1H-benzotriazole-1-yl)-1,1,3, 3 -tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4-methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution and is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and dries in vacuo. Loading 15 capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and dries in vacuo. The 20 final loading capacity of the corresponding solid support 1016 is determined after capping (Prakash et al., J Org. Chem. 2002, 67, 357-369). Example 422 Compounds 1020a (Scheme 1004): The modified nucleoside 1019 is obtained according to literature reports (Tona et al., Org. Lett., 2000, 2, 1693-96). The desired compound 1020a is 25 obtained from compound 1019 by the treatment with triethylsilyl chloride in the presence of imidazole (Oppolzer et al., Helv. Chim. Acta, 1981, 64, 2002). Example 423 Compound 1020b (Scheme 1004): The desired compound 1020b is obtained from compound 1019 according to literature procedures (Chui et al., Bioorg. Med. Chem., 2002, 10, 30 325-332). Example 424 Compound 1021a (X = Me, Scheme 1004): Treatment of compounds 1020a with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient 355 WO 2004/094345 PCT/US2004/011822 temperature in CH 2 C1 2 yield the corresponding phosphoramidite 1021a (Chui et al. J. Org. Chem., 2002, 67, 8847-54). Example 425 Compound 1021b (X = Me, Scheme 1004): Treatment of compounds 1020b with 5 methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2 C1 2 yields the corresponding phosphoramidite 1021b (Chui et al. J. Org. Chem., 2002, 67, 8847-54). Example 426 Compound 1021a (X = allyl, Scheme 1004): To a solution of compound 1020a (1 10 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 102 la (Hayakawa et aL., J Am. Chem. Soc., 1990, 112, 1691-96). Example 427 15 Compound 102ba (X = allyl, Scheme 1004): To a solution of compound 1020b (1 rmmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1021b (Hayakawa et al., J. Am. Chem. Soc., 1990, 112, 1691-96). 20 Example 428 Compound 1021a (X = 0-cyanoethyl, Scheme 1004): The desired phosphoramidite 1021a is prepared from the corresponding precursor 1020a and P-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). 25 Example 429 Compound 1021b (X = P-cyanoethyl, Scheme 1004): The desired phosphoramidite 1021b is prepared from the corresponding precursor 1020b and P3-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357-369). 30 Example 430 Solid supports 1022a-b (Scheme 1004): The desired solid support 1022 is prepared from corresponding precursor 1020 as described in Example 5. 356 WO 2004/094345 PCT/US2004/011822 Example 431 Compounds 1024a-z, 1024al-cl (Scheme 1005): All the compounds (1023a-z and 1023a1-cl) shown in Scheme 1005 are obtained according to reported procedures (Loakers, D.; Nucleic Acids Res., 2001, 29, 2437 or references cited therein). Exocyclic amino group(s) of the 5 heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. The desired silylayted compound 1024 is obtained from the corresponding precursor according to literature procedures (Chui et al., Bioorg. Med. Chem., 2002, 10, 325-332). Example 432 10 Compounds 1025a-z, 1025al-cl (Z= Me, Scheme 1005):Treatment of compounds 1024 with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2

C

2 yield the corresponding phosphoramidite 1025 (Chui et al. J Org. Chem., 2002, 67, 8847-54). Example 433 15 Compounds 1025a-z, 1025al-el (X = allyl, Scheme 1005): To a solution of compound 1024 (1 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamnino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1025 (Hayakawa et al., J Am. Chem. Soc., 1990, 112, 1691-96). 20 Example 434 Compounds 1025a-z, 1025al-cl (X = 3-cyanoethyl, Scheme 1005): The desired phosphoramidite 1025 is prepared from the corresponding precursor 1024 and 3-cyanoethyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J Org. Chem. 2002, 67, 357 25 369). Example 435 Solid supports 1026a-z, 1026a1-cl (R', R" = OSiMe 3 , R"' = OCH(CHs) 2 , Scheme 1005): Compound 1024 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethne 30 (0.9 mL) is added into the mixture, and the mixture is stirred at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtaned is dried over P 2 0 5 under vacuum 357 WO 2004/094345 PCT/US2004/011822 overnight and suspended in anhydrous DMF, mixed with 2 -(1H-benzotriazole-1-yl)-1,1,3,3 tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4 -methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated amount of solid support with an amino tether is added into the clear solution and allows to shake on a shaker at ambient temperature for 18 h. An 5 aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and is dried in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfiunctionalized sites on the support are capped byshaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 10 2 h. The solid support is filtered, washed with CH 3 CN followed by diethylether, and is dried in vacuo. The final loading capacity of the corresponding solid support 1026 is determined after capping (Prakash et al., J. Org. Chem. 2002, 67, 357-369). Example 436 Compounds 1027a-z, 1027al-cl (Scheme 1005): All the compounds (1027a-z and 15 1027a1-cl) shown in Scheme 1005 are obtained according to reported procedures (Loakers, D.; Nucleic Acids Res., 2001, 29, 2437 or references cited therein). Exocyclic amino group(s) of the heterocyclic base is(are) appropriately protected for solid phase oligonucleotide synthesis by following standard protection and deprotection protocols. The desired compound 1027 is obtained from compound 1023 by the treatment with triethylsilyl chloride in the presence of 20 imidazole (Oppolzer et al., Helv. Chim. Acta, 1981, 64, 2002). Example 437 Compounds 1028a-z, 1028a1-cl (Z= Me, Scheme 1005):Treatment of compounds 1027 with methyl tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole at ambient temperature in CH 2

CI

2 yields the corresponding phosphoramidite 1028 (Chui et al. J Org. 25 Chem., 2002, 67, 8847-54). Example 438 Compounds 1028a-z, 1028a1-cl (X = allyl, Scheme 1005): To a solution of compound 1027 (1 mmol) in acetonitrile are added diisopropylamine (1.2 mmol), (allyloxy)bis(diisopropylamino)phospine (1.5 mmol) and 1 H-tetrazole (1.2 mmol). The solution 30 is stirred for 2 h at ambient temperature to obtain the corresponding phosphoramidite 1028 (Hayakawa et al., J Am. Chem. Soc., 1990, 112, 1691-96). Example 439 Compounds 1028a-z, 1028a1-cl (X = P-cyanoethyl, Scheme 1005): The desired phosphoramidite 1028 is prepared from the corresponding precursor 1027 and P3-cyanoethyl 358 WO 2004/094345 PCT/US2004/011822 tetraisopropyl phosphorodiamidite in the presence of 1 H-tetrazole diisopropylammonium salt in anhydrous acetonitrile as reported in the literature (Prakash et al., J. Org. Chem. 2002, 67, 357 369). Example 440 5 Solid supports 1026a-z, 1026a1-cl1 (R', R", R"' = Et, Scheme 1005): Compound 1027 (1 mmol) is mixed with succinic anhydride (2 mmol) and dimethlyaminopyridine (1 mmol), and is dried over P 2 0 5 in vacuo overnight. Dichloromethane (0.9 mL) is added into the mixture, and the mixture is stirred at ambient temperature for 8 h. The reaction mixture is diluted with excess dichloromethane and the organic layer is subjected ice cold aqueous citric acid wash (10 % 10 solution) and brine. The organic phase is dried over anhydrous Na 2

SO

4 and concentrated to dryness to yield the corresponding succinic acid derivative. The succinic acid derivative (1 mmol) thus obtained is dried over P 2 0 5 under vacuum overnight, suspended in anhydrous DMF, mixed with 2-(1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 1 mmol) and 4-methylmorpholine (2 mmol) with vortexing to give a clear solution. Calculated 15 amount of solid support with an amino tether is added into the clear solution, and the mixture is allowed to shake on a shaker at ambient temperature for 18 h. An aliquot of the support is withdrawn and washed with DMF, CH 3 CN and diethylether, and is dried in vacuo. Loading capacity is determined by following standard procedure. Functionalized solid support is then washed with DMF, CH 3 CN, diethylether and dried in vacuo. Unfunctionalized sites on the 20 support are capped by shaking with acetic anhydride/collidine/N-methylimidazole in THF (2 mL Cap A and 2 mL Cap B solutions from Perspective Biosystems Inc.) on a shaker for 2 h. The solid support is filtered, washed with CH 3 CN followed by diethlether, and is dried in vacuo. The final loading capacity of the corresponding solid support 1026 is determined after capping (Prakash et al., J. Org. Chem. 2002, 67, 357-369). 25 Example 441 Synthesis of orthoester reagent and 5'-silyl-2'-ACE-3'-O-(N,N-diisopropylamine) methoxyphosphine-5-methoxyuridine. The reagents in this example can be obtained from a variety of commercial sources, e.g., Aldrich Chemical (Milwaukee, Wis.), TCI America (Portland, Oreg.) and Monomer Sciences 30 (New Market, Ala.). Synthesis of tris(2-acetyl-ethoxy) orthoformate, ACE orthoester reagent: Acetic acid ethyl ester (85%) (5 eq.) is treated with pyridinium p-toluene sulfonate (0.2 eq.) and trimethyl orthoformate (1 eq.). The reaction is heated to distill off the methanol product. The reaction is 359 WO 2004/094345 PCT/US2004/011822 cooled and then neutralized with base. The product is purified by column chromatography and high vacuum distillation. Synthesis of 2 '-O-bis(2-acetyl-ethoxy)methyl 1-methylinosine (representative of general 2'-protection reaction): 5'-O-3'-O-tetraisopropyldisiloxyl 1-methylinosine (TIPS-2-thiouridine, 1 5 eq.) is reacted neat with tris(2-acetyl-ethoxy) orthoformate (2.8 eq.) and pyridinium p-toluene sulfonate (0.2 eq.) at 55.degree. C. for 3 hours under high vacuum (<15 microns of Hg). The reaction is cooled to room temperature and neutralized with base. The crude reaction is passed over silica gel to do a crude purification to remove the neutralized catalyst. The enriched mixture is treated with a premixed solution of N,N,N',N'-tetramethylethylendiamine (TEMED) and 48% 10 hydrofluoric acid in acetonitrile for 6 hours. The product is purified by column chromatography. Synthesis of 5'-O-silyl-2 '-O-ACE-i-methylinosine: To 2'-O-ACE- 1 -methylinosine (1 eq) and imidazole (4 eq.) in tetrahydrofuran was added bis(trimethylsiloxy)-cyclooctoxy silylchloride (OCT-C1) (1.5 eq. in tetrahydrofuran) over 30 minutes with stirring. OCT-C1 can be synthesized by those skilled in the art from bis(trimethylsiloxy)-dichlorosilane and 15 cyclooctanol. The 5'-silyl-2'-ACE 1-methylinosine product is purified by silica gel chromatography. Synthesis of 5 '-0O-silyl-2'-ACE-1-methylinosine-3 '-O-(NN-diisopropylmethoxy) phosphoramidite: To a solution of 5'-O-silyl-2'-O-ACE-l-methylinosine (1 eq.) in dichloromethane is added first bis(N,N-diisopropylamine)methoxy-phosphine (1.3 eq.) followed 20 by tetrazole (0.8 eq.) with stirring. After 2 hours the reaction is quenched and the product isolated via silica gel chromatography. Example 442 Synthesis of Oligonucleotides Oligonucleotide synthesis can be conducted e.g., on a Gene Assemble Plus synthesizer 25 from Pharmacia of Milwaukee, WI or a 380 synthesizer (ABI). The protocols can be adapted by those skilled in the art to any commercially available synthesizer. A solid support, e.g., a derivatized polymer support can be used for all syntheses. and includs a thymidine polystyrene support with succinate linker from Pharmacia packed in 0.2 or 1.0 .mu.mole columns purchased form Miligen of Milford, Mass. The silyl deprotection reagent can be carried out as follows: 30 1.0M aqueous HF solution (Mallinkrodt) and 1.6M triethylamine ("TEA") solution in dimethylformamide (DMF). Wash solvents are acetonitrile ("MeCN") or DMF and are used between the reactions. Amidites were dissolved to 0.1M in acetonitrile. The coupling catalyst is 0.15M S-ethyl-tetrazole for RNA synthesis. Capping solutions include commercially available standards of acetic anhydride and N-methyl imidazole or dimethylaminopyridine. Oxidation was 360 WO 2004/094345 PCT/US2004/011822 effected during every cycle using 3 M t-butylhydroperoxide (tBuOOH) in toluene. Table 2 includes an outline of the oligonucleotide synthesis cycle conditions. Table 2 Reaction Reagent Time (Seconds) 5 5'-silyl deprotection, l.0 M HF & 1.6 M TEA in DMF, 30-35 Wash DMF 10 Wash MeCN 40 Couple 15 eq. amidite/100 eq. S- 90 ethyl-tetrazole 10 Wash MeCN 30 Oxidize tBuOOH 40 Wash MeCN 30 Capping 10% acetic anhydride & 10% 30 N-Methyl imidazole 15 Wash MeCN 30 Wash DMF 5 Following synthesis on the synthesizer, the polymer support is treated for 30 minutes using a 1 M solution of disodium-2-cobamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMF. The 20 S 2 Na 2 reagent is washed out with water and acetone. The dried support is treated with 1 ml of 40% N-methylamine in water for 10 minutes at 55.degree. C. to cleave all base-labile protecting groups and release the 2'-protected oligonucleotide into solution. The crude reaction mixture is analyzed by anion exchange high pressure liquid chromatography (HPLC) and is dried down in vacuo. The pellet is resuspended in 1.6 ml of 50 mM acetic acid, pH 3.0, and incubated for 10 25 minutes at 55.degree. C. To this is added 1.6 ml of 150 mM TRIS, pH 8.7, (Final pH of solution 7.7-8.0) for 10 minutes at 55.degree. C. An aliquot of this solution is then analyzed by essentially the same HPLC conditions. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope 30 . of the invention. Accordingly, other embodiments are within the scope of the following claims. 361

Claims

1. A protected monomer having a formula (1) X 5' X5"-S I-O X Si O--- B 1 ' o X 3 X2 (I) 5 wherein, B is selected from the group consisting of: 362 WO 2004/094345 PCT/US2004/011822 R' R 2 R 9 N R 4 R 4 ' 7 1 0 N R 8 R R N R 2 N / N R\N N Ri Ri12 R3 N R N N NX / N N R 14 N N,1 N N NR N N R Y O NH R 19 '1,NR20 21 R 24 , -R25N R l R Rq 230 N NH X N 26R2 O N | R 22 anthracenyl, pyrenyl, 5 10 363 WO 2004/094345 PCT/US2004/011822 R53 R 54 R46 Rs51 Q Q~ Q, Qiv! 52 QiV Rss IIR 2K Q N 'N R48 Rse6 I R62 O R 61 R63 , I iv Q , and R6 0 R 57 R64 R67 R 59 R58 R6 5 R 66 R72 1, iv Q R68 71 R R7o R69 X 2 is an ortho ester protecting group, hydrogen, ethers, alkyl ethers, esters, halogens, protected amines, or protected hydroxyl moieties; 5 X is -O-P(OR 27 )N(R 28 ) 2 or -O-L-R2; X', Xs", X s "' include at least one alkoxy or siloxy substituent; R1 is hydrogen or CI-C 4 alkyl; R 2 is hydrogen, CI-C 4 alkyl, or C 2 -C 6 alkenyl optionally substituted with hydroxy, or C(O)NHRa; 10 R 3 is hydrogen, halo, Ci-C 4 alkyl, C 1 -C 4 thioalkoxy, NH 2 , NHR b, or NRbRC; 364 WO 2004/094345 PCT/US2004/011822 R 4 when taken together with R 4' forms oxo, or R 4 when taken together with R 5 forms a double bond between the carbon and nitrogen atoms to which they are attached; R 4' when taken together with R 4 forms oxo, or is O'; R 5 is hydrogen, CI-C 4 alkyl, or when taken together with R 4 forms a double bond 5 between the carbon and nitrogen atoms to which they are attached; R 6 is hydrogen, halo, NH 2 , NHR b , or NRbR; R 7 is an unshared electron pair, or C 1 -C 4 alkyl; R8 when taken together with R 9 forms a double bond between the carbon and nitrogen atoms to which they are attached, or R8 when taken together with R" 1 forms a double bond 10 between the carbon and nitrogen atoms to which they are attached; R 9 is hydrogen, CI-C 4 alkyl, or when taken together with R 8 forms a double bond between the carbon and nitrogen atoms to which they are attached; R 1 0 is hydrogen or is absent; R 1 is hydrogen, CI-C 4 alkyl, or when taken together with R8 forms a double bond 15 between the carbon and nitrogen atoms to which they are attached; R12 is hydrogen, formyl, or C 1 -C 4 alkyl optionally substituted with hydroxy or protected hydroxy; R 13 and R 14 are each independently hydrogen or C 1 -C 4 alkyl; R 1 5 is hydrogen, C 1 -C 4 alkyl, or (CH 2 )nCH(Rd)CH(NHRe)(COOR); 20 R 16 is hydrogen or C 1 -C 4 alkyl; R 17 is halo, NH 2 , NHR b, or NRbR; R 1 8 is cyano, C(=NH)NH 2 , or CH 2 NH(Rh); R1 9 is hydrogen, or C 1 -C 4 alkyl; R 20 is: 25 (i) hydrogen; (ii) hydroxy or protected hydroxy; (iii) C 1 -C 4 alkoxy optionally substituted with COOR'; or (iv) CI-C 4 alkyl optionally substituted with hydroxy and/or COOR, NH 2 , NI t m , or CONH2; 30 R 2 1 is hydrogen, or when taken together with R 23 forms a double bond between the carbon atoms to which they are attached; R 22 is hydrogen; R23 is hydrogen, or when taken together with R 2 1 forms a double bond between the carbon atoms to which they are attached; 365 WO 2004/094345 PCT/US2004/011822 R24 and R 25 are each, independently, hydrogen or C 1 -C 4 alkyl; R 26 is (CH 2 )nCH(Rd)CH(NIIRe)(COOR); R2 7 is C 1 -C 6 alkyl optionally substituted with cyano, or C 2 -C 6 alkenyl; R 28 is C 1 -Clo alkyl; 5 R 29 is a liquid or solid phase support reagent; Q is N or CR 44 ; Q' is N or CR 45 ; Q" is N or CR 47 ; Q"' is N or CR 49 ; 10 QiV is N or CR 5 s; R 44 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR, C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 forms -OCH 2 0-; R 45 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NRbRc, C 1 -C 6 15 alkyl, C 6 -C 10 aryl, C 6 -C 0 lo heteroaryl, C 3 -Cs heterocyclyl, a ligand, a tethered ligand, or when taken together with R 44 or R 46 forms -OCH 2 0-; R 4 6 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR c , Ci-C 6 alkyl, C 6 -Co 10 aryl, C 6 -C 1 0 heteroaryl, C 3 -C 8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 45 or R 47 forms -OCH20-; 20 R 4 7 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NI b , or NRbR , C 1 -C 6 alkyl, C 6 -Co 10 aryl, C 6 -C 1 0 heteroaryl, C 3 -C 8 heterocyclyl, a ligand, a tethered ligand, or when taken together with R 4 6 or R 4 8 forms -OCH20-; R 48 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH2, NIRb , or NRbR e, C i-C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, a ligand, a tethered ligand, or when 25 taken together with R 47 forms -OCH 2 0-; R 4 9 RSO, R 5 1 , R 5 2 , R 5 3 , R 5 4 , R 5 7 , R 5 8 , R 5 9 , R 6 0 , R 6 1 , R 6 2 , R 6 3 , R 64 , R 6 5 , R 6 6 , R 6 7 , R 6 8 , R 6 9 , R 7 0, R 71, and R 72 are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR, C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -Co 10 aryl, C 6 -Co 10 heteroaryl, C 3 C 8 heterocyclyl, NC(O)R 7 , or NC(O)R; 30 R 55 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHRb, or NRbR e, CI-C 6 alkyl, C 2 -C 6 alkynlyl, C 6 -C 10 aryl, C 6 -C 1 0 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 7 , or NC(O)Ro, or when taken together with R 5 6 forms a fused aromatic ring which may be optionally substituted; 366 WO 2004/094345 PCT/US2004/011822 R 56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NII b , or NRbR , C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 1 0 aryl, C 6 -Co 10 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 7 , or NC(O)Ro, or when taken together with R 55 forms a fused aromatic ring which may be optionally substituted; 5 X is O, S, or Se; Yis O orS; L is -C(O)(CH 2 )qC(0)-, or -C(O)(CH 2 )qS-; Provided that R 1, R2, and R 3 cannot all be hydrogen; further provided that when R 5 is hydrogen, R 6 cannot be NH 2 , NiH(protecting group), or NH(iBu); further provided that when R12 10 is hydrogen and R 8 and R 11 together form a double bond between the carbon and nitrogen atoms to which they are attached, R 9 and R 1 0 cannot both be hydrogen; further provided that when X and Y are O, R 1 9 is hydrogen, and R 21 and R 23 together form a double bond between the carbon atoms to which they are attached, R 2 0 cannot be hydrogen or CH 3 ; Ra is glycinyl, threonyl, or norvalyl, each of which may optionally be partially or fully 15 protected; R b is C 1 -C 6 alkyl or a nitrogen protecting group; Re is Ci-C 6 alkyl; R d is hydrogen, hydroxy, protected hydroxy, or OOH; Re is hydrogen, a nitrogen protecting group, or COOR; 20 R f is hydrogen, or C 1 -C 6 alkyl; Rg is CI-Co 10 alkyl; R is hydrogen, or Ri RkO Ri RIO R' and Rj when taken together forms a double bond between the carbon atoms to which 25 they are attached, or R' and Rj when taken together form -0- between the carbon atoms to which they are attached; 367 WO 2004/094345 PCT/US2004/011822 R k and R are each, independently, hydrogen, a hydroxyl protecting group,a sugar, or a fully or partially protected sugar; R m is C 1 -C 4 alkyl optionally substituted with COOH; R' is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH 2 , 5 NHR b , or NRbR , CI-C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 6 -C 1 0 heteroaryl, C 3 -Cs heterocyclyl, NC(O)R 17 , or NC(O)Ro; n is 1-4; and q is 0-4. 368 WO 2004/094345 PCT/US2004/011822

2. The monomer of claim 1, wherein B is: R1 N R 2 R\ N N R3 N 5

3. The monomer of claim 1, wherein B is: R 4 R4' R Rs 5 NN N N R 6 N

4. The monomer of claim 1, wherein B is: R 9 RiO-N R R R 12 N X N 10

5. The monomer of claim 1, wherein B is: 369 WO 2004/094345 PCT/US2004/011822 R, O R15 0 R14 N!N N'I'N N) R 13

6. The monomer of claim 1, wherein B is: 5 0 R18 O R N N RN 17 N

7. The monomer of claim 1, wherein B is: Y 20 R N9'1 R 21 R23 R X N I R 22 10 370 WO 2004/094345 PCT/US2004/011822

8. The monomer of claim 1, wherein B is: O R 24 _ R 2 5 Oy 5

9. The monomer of claim 1, wherein B is: NH 1 I

10. The monomer of claim 1, wherein B is: R46 R48 Ii 10 371 WO 2004/094345 PCT/US2004/011822

11. The monomer of claim 1, wherein B is: Ri Q N 5

12. The monomer of claim 1, wherein B is: R 53 R 5 4 Q oiv// R5 N R 5 6

13. The monomer of claim 1, wherein B is: R62 0 R61 R60 R57 59 R5 10 372 WO 2004/094345 PCT/US2004/011822

14. The monomer of claim 1, wherein B is: R64R 6 7 R 63 ,,, Q iv,, -Q S5 R ,65 R66 B 5

15. The monomer of claim 1, wherein B is: R 72 Qy iv Q R 68 R 71 R 70 R69

16. The monomer of claim 1, wherein B is anthracenyl. 10

17. The monomer of claim 1, wherein B is pyrenyl.

18. The monomer of claiml, wherein R 28 is isopropyl. 15

19. The monomer of claim 1, wherein X 5 ', X 5 ", and X 5 ' are any combination of the following formula: 373 WO 2004/094345 PCT/US2004/011822 003C OH -O- OH HC-Si OS PAPJ CH3 3 3 3 3 5

20. The compound of claim 1, wherein X 5 ' and X s' are siloxy and X 5 "' is cycloalkoxy. 374 WO 2004/094345 PCT/US2004/011822

21. The monomer of claim 1, wherein the orthoester protecting group has a formula(III): O R320 OR31 (Il) 5

22. The monomer of claim 21, wherein R 31 and R 32 are the same or different and are any combination of the following formulae: 10 375 WO 2004/094345 PCT/US2004/011822 C, Cl OCH 3 CI CNO CI Cl CI CN R 3 3 R 34 R 3 6 R33 O R 34 R33 R3 37 RR35 O R38 38 O 0 R 3 6 R3 orO or 0 ,,NO R38 wherein R 33 , R 34 , R 35 , R 3 6, and R 37 is a compatible ligand, or hydrogen, or halogen, alkyl, or cyano substituent, and R 38 is compatible ligand. 5 376 WO 2004/094345 PCT/US2004/011822

23. The monomer of claim 21, wherein the orthoester is: H 3 C O O O CH3 o O 5

24. The monomer of claim 1, wherein R 29 is a fluoride-stable polystyrene based solid support or PEG.

25. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(0)CH3] 2 ; R 27 is CH3; 10 R2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: R N R 2 N N R 3 N R

26. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH20C(O)CH 3 ] 2 ; R 27 is CH3; R 2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is: 15 R 4 R 4' R R5 NN R6 N 377 WO 2004/094345 PCT/US2004/011822

27. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 0C(O)CH3] 2 ; R 2 7 is CH 3 ; R28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is R 9 R 1 0 -N R 8 R 1 1 R 12 N X N 5 378 WO 2004/094345 PCT/US2004/011822

28. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH 3 ] 2 ; R 27 is CH3; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: R 15 0 R N N NN R 13 5

29. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH20C(0)CH 3 ] 2 ; R 27 is CH 3 ; R 2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: 0 R18 OR R16 R"N N 10

30. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH 3 ] 2 ; R 27 is CH 3 ; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is: Y 19 R20 R N RR21 R23 X N I R

31. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(0)CH 3 ] 2 ; R 27 is CH3; 15 R 2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: 379 WO 2004/094345 PCT/US2004/011822 0 R 24 I R 25 O

32. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(0)CH 3 ] 2 ; R 27 is CH 3 , R2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: NH R26 NI I R 2HN N 5

33. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 0C(0)CH 3 ]2; R 27 is CH3; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: R46 Q' Q II Q R 48 10 380 WO 2004/094345 PCT/US2004/011822

34. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH 3 ] 2 ; R 2 7 is CH 3 ; R 2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: R 51 Q Qiv 52 N 5

35. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH3]2; R 27 is CH 3 ; R 2 8 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is: R53 R 5 4 Qi t iv R155 'N R56 381 WO 2004/094345 PCT/US2004/011822

36. The monomer of claim 1, wherein X 2 is -OC[OCH2CH 2 OC(O)CH 3 ] 2 ; R 27 is CH 3 ; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: 5 R 6 2 O R61 R 6o R57 R 59 R ea

37. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH 3 ] 2 ; R 2 7 is CH 3 ; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is: R63 ,,Q ivQ R64 6 7 R65 R66 10

38. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH20C(O)CH3] 2 ; R 27 is CH 3 ; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is: R72 ,Q iv Q R68 R 7 1 R 70 R 69 15 382 WO 2004/094345 PCT/US2004/011822

39. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH2OC(O)CH3] 2 ; R 2 7 is CH 3 ; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is anthracenyl. 5

40. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH3] 2 ; R 27 is CH3; R 28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5"' is cyclododecyloxy; and B is pyrenyl.

41. The monomer of claim 1, wherein B is selected from the group consisting of: 2-aminoadeninyl 10 2-methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl, N6-isopentenyladeninyl, 2 -methylthio-N6-isopentenyladeninyl, 15 N 6 -(cis-hydroxyisopentenyl)adeninyl, 2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl, N6-glycinylcarbamoyladeninyl, N6-threonylcarbamoyladeninyl, 2-methylthio-N6-threonyl carbamoyladeninyl, 20 N 6 -methyl-N6-threonylcarbamoyladeninyl, N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methylcytosinyl, 25 5-methylcytosinyl, 2-thiocytosinyl, 5-formyleytosinyl, NH COOH N H 2 N N N H N4-methylcytosinyl, 30 5-hydroxymethylcytosinyl, 1-methylguaninyl, 383 WO 2004/094345 PCTIUS2004/01 1822 N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl, 5 384 WO 2004/094345 PCT/US2004/011822 NHCOOCH 3 NHCOOCH 3 NHCOOCH 3 H 3 COOC H 3 COOC OH H 3 COOC OOH OOO 0 0 N N, N N) N H~3C H 3 C 1 N HC N N N N N N N CH 3 CH 3 CH, NH 2 HOOC 'OH O OH 3 C 0 0 -N-'> N /NO N H3C;G H3C\ H 3 N N N N H N /N N H. /- N N , CH 3 CH 3 CH 3 O 0 HO HO HN' N H HC0 beta-galactosyl O O beta-mannosyl O HO SNH H 2 N H 2 0 NH2 5 N2,7-dimethylguaninyl, N2,N2,7-trimethylguaninyl, 1-methylguaninyl, 10 7-cyano-7-deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl, 385 WO 2004/094345 PCTIUS2004/01 1822 dihydrouracilyl, 5-methyluracilyl, 1 -methylpseudouracilyl, 2-thiouracilyl, 5 4-thiouracilyl, 5-methyl-2-thiouracilyl, 3-(3-amino-3-carboxypropyl)uracilyl, 5-hydroxyuracilyl, 5-methoxyaracilyl, 10 uracilyl 5-oxyacetic acid, uracilyl 5-oxyacetic acid methyl ester, 5-(carboxyhydroxymethyl)uracilyl, 5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonylmethyluracilyl, 15 5-methoxycarbonylmethyl-2-thiouracilyl, 5-aminomethyl-2-thiouracilyl, 5-methylaminomethyluracilyl, 5-methylaminomethyl-2-thiouracilyl, 5-methylamninomethyl-2-selcnouracilyl, 20 5-carbamoylmethyluracilyl, 5-carboxymethylaminomethyluracilyl, 5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl, 1 -methyl-3-(3 -amino-3-carboxypropyl) pseudouracilyl, 25 5-carboxymethyluracilyl, 5-methyldihydrouracilyl, 3-methyipseudouracilyl, H F C 3 NH 2 NH 2 0 2 N H 3 C N N NN I F & 386 WO 2004/094345 PCT/US2004/011822 NO 2 , BuHN N N0 N NN N HI N- ON O H H N - 2 CH 3 0 CH 3 O O CH 3 3 HsC CH 3 0 CH 3 H 3 ~CH 3 N NN ') N" N - -CH 3 CH 3 , , and N CH 3 5

42. The monomer of claim 1, wherein X 2 is -OC[OCH 2 CH 2 OC(O)CH 3 ] 2 ; R 27 is CH 3 ; R28 is (CH 3 ) 2 CH-; X5' and X5" are trimethylsiloxy; X5'" is cyclododecyloxy; and B is selected from the group consisting of: 2-aminoadeninyl, 2-methyladeninyl, 10 N6-methyladeninyl, 2-methylthio-N6-methyladeninyl, N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl, N6-(cis-hydroxyisopentenyl)adeninyl, 15 2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl, N6-glycinylcarbamoyladeninyl, N6-threonylcarbamoyladeninyl, 2-methylthio-N6-threonyl carbamoyladeninyl, N6-methyl-N6-threonylcarbamoyladeninyl, 387 WO 2004/094345 PCTIUS2004/01 1822 N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6--hydroxynorvalyl carbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methyleytosinyl, 5 5-methylcytosinyl, 2-thiocytosinyl, 5-formylcytosinyl, NH COOH N, H 2 N N N HI N4-methyleytosinyl, 10 5-hydroxymethylcytosinyl, 1 -methylguaninyl, N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl, 15 NHCOOCH 3 NHOOOCH 3 NHCOOCH 3 H 3 0000 H 3 0000 'OH H 3 COOC 'OOH 0 0 0 H 3 0 C N H 3 ~ N- N 3 NAN N N NN N OH 3 6H 3 OH, NH 2 HOOC 'OH 0 H 3 C 0 / N N N N H 3 0 N< 3 N N H N N N N N N H, N 6H3 388 WO 2004/094345 PCT/US2004/011822 N2,7-dimethylguaninyl, N2,N2,7-trimethylguaninyl, 1-methylguaninyl, 5 7-cyano-7-deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl, dihydrouracilyl, 5-methyluracilyl, 10 1-methylpseudouracilyl, 2-thiouracilyl, 4-thiouracilyl O 0 H H HN N H 3 C\N ~ o %._I , , NN HO O HO NN N N ' 0 N O NH NH HN HN H 2 N N H 2 N N HO H beta-galactosylO O beta-mannosyl 0 O 0 0 0HN O NH 0 NH 0 NH 2 HN ,> HN HN H 2 N"1N N H 2 N N N H 2 N NN 5-methyl-2-thiouracilyl, 3 -( 3 -amino-3-carboxypropyl)uracilyl, 15 5-hydroxyuracilyl, 5-methoxyuracilyl, uracilyl 5-oxyacetic acid, uracilyl 5-oxyacetic acid methyl ester, 5-(carboxyhydroxymnethyl)uracilyl, 20 5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonylmethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl, 389 WO 2004/094345 PCTIUS2004/01 1822 5-aminomethyl-2-thiouracilyl, 5-methylatminomethyluracilyl, 5-methylamninomethyl-2-thiouracilyl, 5-methylaminomethyl-2-selenouracilyl, 5 5-carbamnoylmethyluracilyl, 5-carboxymethylamninomethyluracilyl, 5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl, 1 -methyl-3 -(3-amino-3-carboxypropyl) pseudouracilyl, 10 5-carboxymethyluracilyl, 5-methyldihydrouracilyl, 3-mecthylpseudouracilyl, F H 3 NH 2 NH 2 0N H 3 C Al 02NN~ F N0 2 0 ~ /N ,BuHN'k N K(X. " N N--[, H N - 0 2 N 0 H OH 3 0 OH 3 0 0O H 3 OH 3 H 3 C3 H3 3 N< ~ j 0- - OH H3 OH 3 N OH 3 15 390 WO 2004/094345 PCT/US2004/011822

43. The monomer of claim 1, wherein X 2 is fluoro.

44. The monomer of claim 1, wherein B is: F CH 3 F 5

45. The monomer of claim 1, wherein B is substituted or unsubstituted aryl attached to a tethered or untethered ligand. 10

46. A protected monomer having a formula: ------ u(H 2 C) N S in which u is 1 or 2; the wavy line represents a point of attachment for a ligand or a tethered ligand; and the dotted lines represent points of attachment for a first functionalized hydroxyl 15 group; a second functionalized hydroxyl group; and an unfunctionalized hydroxyl group, a protected hydroxyl group, or hydrogen. 391 WO 2004/094345 PCT/US2004/011822

47. The monomer of claim 46, wherein the first functionalized hydroxyl group has the formula: X5 ' Xs"-Si-O x5" 5 ; in which X 5 " , X 5' , and X 5' . include at least one alkoxy or siloxy substituent.

48. The monomer of claim 46, wherein the second functionalized hydroxyl group has one of the following formulas: I or (R28) 2 N OR 2 7 L 10 ; in which R 27 is C 1 -C 6 alkyl optionally substituted with cyano or C 2 -C 6 alkenyl; R 28 is C 1 -C 10 alkyl; * is a solid or liquid support reagent; and L is a linker. 15

49. The monomer of claim 46, wherein the ligand is a targeting group.

50. The monomer of claim 49, wherein the targeting group is a lipid, steroid, vitamin, carbohydrate, polyamine, amino acid, peptide, peptide mimetic or cleaving molecule. 20 392 WO 2004/094345 PCT/US2004/011822

51. The monomer of claim 50, wherein the steroid is cholesterol.

52. The monomer of claim 46, wherein the ligand is a diagnostic group. 5

53. The monomer of claim 52, wherein the diagnostic group is biotin, a fluorophore, an antibody or an antigen.

54. The monomer of claim 46, wherein the ligand has a formula (G)C(=H)NHR, in which G is -0-, -NH-, or -CH 2 -; H is O or NH; and R" is H, CI-C 6 alkyl, C 6 -Clo aryl, or C 5 -Co 0 10 heteroaryl.

55. The monomer of claim 46, wherein the monomer has a tethered ligand.

56. The monomer of claim 55, wherein the ligand is tethered with a tether selected from 15 the group consisting of: -C(0)-(CH 2 )s-C(0)-(ligand); -C(O)-(CH 2 ),-C(0)O-(ligand); -C(0)-O (ligand); -C(O)-(CH2)s-NH-; -C(O)-(CH2)s-NH-C(O)-(ligand); -C(O)-(CH2)s-(ligand); -C(O) NH-(ligand); -C(O)-(ligand); -(CH 2 )s-C(O)-(ligand); -(CH 2 )s-C(O)O-(ligand); -(CH 2 )s-(ligand); (CH 2 )s-NH-; and -(CH 2 )s-NH-C(0)-(ligand), wherein s is 0-6. 20 393 WO 2004/094345 PCT/US2004/011822

57. The monomer of claim 46, wherein the monomer has the formula: 0 X5,N O-chol O-P-N(ipr) 2 I H-p OCH 2 CH 2 CN or 00 X 5, O H N O-chol X 5 'I H O-C(0)CH2CH2C()NH H-ss 5 wherein, X 5 ', X s 5 " ' , and X 5 "' include at least one alkoxy or siloxy substituent, ipr is an isopropyl group, and chol is a cholesterol radical.

58. An iRNA agent having a monomer of claim 1 or 46. 10

59. A method of making an iRNA agent, the method comprising providing an iRNA agent having a monomer of claim 1 or 46 and allowing it to anneal to a complementary RNA sequence to form an iRNA agent. 394