CA2343067A1 - Hairpin hybridizer molecules for modulation of gene expression - Google Patents

Abstract

The invention features novel hairpin hybridizer nucleic acid molecules which are able to modulate gene expression; useful in target validation, gene function identification and in human therapeutics.

Description

wo oon» Pcrnrs~ms6s D~~RIPTION
HAIRPI~1 HYBRIDIZER M, LQ ~CUt~~ ~QR MODULATTON OF GENE
s 'Ibis patent application claims the belt of Hartmann et al., USSN
60/101,174, filed September 21, 1998 entitled "HAIRPIN HYBR1D1ZER MOLECULES FOR
MODULATION OF GENE EXPRESSION." This application is hereby incorporated by reference herein in its entirety including the drawings.
Hac c~nd of the Invention l o This invention relates to nucleic acid molecules, which the Applicant terms "hairpin hybridizer" (HPH) molecules that are capable of modulating gene expression by hybridizing to target RNA with improved specificity to thereby block translation of such target RNA.
The following is a discussion of relevant art, none of which is admitted to be l a prior art to the present invention.
Since the discovery of'the mechanisms underlying gene expression, specifically nucleic acid based transcription and translation, a great deal of effort has boett placed on blocking or altering these processes for a variety of purposes, such as understanding biology, gem function, disease processes, and identifying novel therapeutic targets.
zo Approaches involving the use of nucleic acid molecules for modulating gene expression have gained popularity in recent years. For example, nucleic acid molecules have been designed which are capable of binding to'pecific mRNA sequences by Watson-Crick base-pairing interaction and blocking translation (Crooke, 1996, Medicirurl Res. Rev.
16, 319-344). Another approach involves complexation of DNA with triplex-forming wo eon~3ls rcrNS~ams oligonucleotides to prevent transcription of bound DNA sequences thereby inhibiting gene expression (Kim et al., 1998, Biochemistry. 37, 2299-2304). The interaction of antisense oligonucleotides, 2-SA antisense chimera, or ribozymes with target RNICs have been used to modulate gene expression. All of these nucleic acid molecules are s highly specific to their matching target sequences and therefore may offer lower toxicity compared to traditional approaches such as chemotherapy.
The concept of gene expression inhibition through an antisense mechanism is derived in part from meclumis~ns found in nature. It was found that prokaryotic systems utilized complementary RNA molecules to inhibit translation (Lacetena et al., ~ 0 1983, Blood 170, 635-650). Simons et al., 1983, Cell 34, 673-682; Mizuno et al., 1984, Proc. Natl. Aced Sci. (USA) 81, 1966-1970). For example, expression of E. coli OmpF
and OmpC genes (outer membrane proteins} is regulated by an antisense RNA
mechanism (Mukopadhyay Bc Roth, 1996, Critical Rev In Oncogenesls ?,151-190}.
Antisense oligonucleotides can be used to down-regulate target mRNA by a i s number of different mechanisms. The specificity of these reagents is determined by the primary sequence (GC content, sequence length), chemistry (ribonucleotides, deoxy-ribonucleotides, chemically modified nucleotides) of the antisense molecule and the presence of pseudo-target sequences. Pseudo-targets are nucleic acid sequences, which may have sequence identity or homology to a target sequence. The number of pseudo-Zo targets for a given sequence, espocially human genes, is largely unknown at this point, since only a minor fraction of the human genome is currently sequenced.
Lizardi et al., US Patent No. 5,312,728, describe a self hybridizing nucleic acid molecule, referred to as a molecular switch used, for the detection of target nucleic acid molecules. The molecular switch consists of a probe sequence of 20 to 60 nucleotides 25 capable of hybridizing to a target sequence and 5' and 3' sequences of at least 10 nucleotides in length which are capable of hybridizing to each other intramolxularly.
Bngdahl et al., 1997, Nucleic Acids Research 25, 3218-3227, describe the use of an RNA cassette system for silencing the lacl gene. The molecules used consisted of a we oonr~s Pc~rnrs~mass hairpin structure, which was used for target sequence recognition and an inhibitor region which was either an artrxsense or ribozyme sequence.
Dclibas et al,, 1997, Natwx Biotech 1 S, 751-753, gibe the formation of non=
canonical base-pairs using natural satisense RNA and target RNA.
Stinchcomb et al., International PCT Publication NO. WO 95/23225, describe an RNA molecatle with an itrasmtolocular stem loop structure of greater tinm or equal to Wight base_pairs.
This invention relates to micleic acid molecules capable of binding and blocking ~ o the fimaron of target nucleic acid molecules, thereby modulating cellular or viral mechanisms including splicing, editing, replication or gene expression, and translation.
Specifically, the invention concerns novel nucleic acid molecules with a hairpin-secondary st<ucdu~a capable of down regulating protein expression by binding (static biocker) and optionally facilitating the cleavage of target RNA through as RNase H or l s other mechanism. For simplicity and ease of understanding the instant invention, the nucleic acid molecules of the instant invention shall be referred to as hairpin hybridi~er (HPH) molecules. In particular, applicant describes the use of these HPH
molecules to down-regulate gene expression in bacterial, microbial, fungal, eukaryotic systems including plant, or mammalian cells. Down-regulation of specific target sequences may zo either have a therapeutic effect is many diseases or disease states or aid in the identificartion of gene function and/or new therapeutic gene targets. The HPH
molecules of the present invention can be used for in vitro or in vivo applications well known in the art.
The present invention feauues a method of modulating the function of a target xe sequence in a cell using HPH molecules. HPH molecules include target-binding region and a hairpin region, where the target binding region is capable of binding to the target wo oon~3ds pcrrus~aisss sequence in a sequence specific manner in vitro or in vivo to modulate the function of the target sequence. The hairpin region of the HPH molecule provides an improved specificity characteristic to the HPH molecule. The hairpin region is expected Fo provide improved resistance to nuclease degradation, is expexted to help tlx HPH
s molecule with localization inside a cell, and is expectod to help in improved uptake of the HPH molecule by the cells compared to a molecule tacking such a hairpin st<ucr<u~e.
The target binding region of the HPH molecule may also include an RNase H-activating region where such a region includes a greater than or equal to 4 deoxyribonucleotide nucleotide sequence with phosphorothioate, pl~hodiester, i o phosphorodithioate, arabino, 2'-fluoro arabino-and/or S'thiophosphate intemucleotide linkages. The RNase H-activating region interacts with the target RNA to form a DNA:RNA complex which is recognized by the cellular RNase H enzyme, which binds the DNA:RNA complex and cleaves the RNA portion of the DNA:RNA complex.
Such cleavage of the target RNA by RNase H causes the target RNA to lose its normal i a function by causing inhibition of its translation into proteins, its replication, its packaging into viral particles, or other functions.
Thus in a first aspect, a method of modulating the function, such as expression, of a target sequence, preferably in a cell, comprising the step of contacting said target sequence with a HPH' nucleic acid molecule under conditions suitable for the zo modulation of said target sequence expression, wl~ein the HPH nucleic acid molocule includes the following formulae:

wo oom~s rcrros~mass s Formula I:
B B' (~)r ~~)f (~i . ~l~b (P)k ~P)t ~ (M~
where, each P, Y, N and M represents in~lependeatly a nucleotide which may be s the same or different; ~ indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; o is an integer greater than or equal to 3, more specifically 4, 5, 6, ?, 8 or 9; k is zero or an integer greater than or equal to 3 and preferably less than about I00, more specifically 4, 5, 6, ?, 8, 9, I0, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, f o more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 s, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, ?, 8, 9, 10, 11, 12, 13, 14, 15, or 20; (P), and (P~, are independently oligonucleotides, preferably including at least one position that is not deoxynucleotide (e.g. 2'-H containing nucleotide); (P~ and (P~, may include phosphodiester, phosphorothioate, phosphorodithioate, f s methylphosphoaete linkers and the like or a combination thereof; k sad t may be the same length (kat) or different lengths (k ~ t); (M~" is an oligonucleotide sequence whose miter-nucleotide linkers include plmsphodiester, phosphorothioate, S'thiophosphate, or methylp>msphonate linkers or a combination thereof; t, k, and w may be of the same length (k = t = w) or different length {k ~ t ~ w) or (k =
t ~ w) or (k Zo -~ t = w) or (k = w ~ t); at least one or more of of each said (P~, (P)k, and (M)"" is as wo oort~~s rcrnrs~niass oligonucleotide of su~cient length to stably interact independently with a target sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); r and f are independently an integer greater than or equal to zero, specifically 1, 2, 3, 4, 5, 10, or 15; tech B and B' independently represents a cap structure which may independently s be present or absent; when r= 0, and f=0, B and/or B'; when present, is attached to (N~N')~; and repraaits a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, plwsphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).
l o Formula II:
(M~1N
(P)k (P)t IP)k1 (P~t1 (M~
where, each P, N, and M represents independently a micleotide which may be same or differem; ~ indicates hydrogen bond forniarion between two adjacent l s nucleotides, N' is a nucleotide complementary to N; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero'or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 S, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; k1 is zero or as integer greater zo than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, wo oon~~s Pcrnrss9nts6s 10, 11, 12, 15, or 20; tl is zero or an integer greater than or equal to 3 and prefentbiy less than about 100, more specifically 4, S, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than t00, more specifically 5, 6,'7, 8, 9, 10, 11, I2, 13, 14, 15, or 20; (Ph and (P~ , (P~,, and (P~, are independently oligomicleoddes, preferably including at least one position that is not deoxynucleotide (e.g. 2'-H containing nucleotide); (P), and (P~" (P)", (P~, may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination theroof; k and t may be the same length (k~) or differeut lengths (k * t); kl and tl may be the same length (kl=tl) or different lengths (k * t);
i o (M~" is an oligonucleotide sequence whose inter-nucleotide linkers include phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate, or phosphorodithioate linkers or a combination thereof; t, k, and w may be of the same length (k = t = w) or different length (k * t * w) or (k = t * w) or (k * t =
w) or (k = w t); tl, kl, and w may be of the samc length (kl = tl= w) or different length (kl * tl m w) or (kl = tl * w) or (kl * tl = w) or (kl = w * tl); at least one or more of each said (P~, (P~, (P)", (P~, and (Ivnw is an oligonucleotide of sufficient length to stably inten3ct independently with a target sequence (the target can be an RNA, DNA or RNA/DNA
mixed polymers); and represarts a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'-thiophosphate, 2o methylphosphonate, or others known in the art).
Formula III:

wo oonr~sb Pcrrms~mass (M)w (P)k (~It (N~N)o D \E
i i B B' where, each P, N, and M represents independently a nucleotide which may be same or different; ~ indicates hydrogen bond fonmation between two adjacxnt s nucleotides, N' is a nucleotide complementary to N; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, S, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or ~ o equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; (P)< and (P~ are oligonucleotides preferably including at toast one p~ition that is not deoxynacleotide (e.g. 2'-H containing nucleotide); each(P~
and (P~
may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; k and t may be the i s same length (k=t) or different lengths (k ~t); (M)W is an oligonucleotide sequence whose inter-nucleotide linkers may include phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate, or phosphorodithioate linkers or a combination thereof; t, k, and w may be of the same length (k = t = w) or different length (k x t ~ w) or (k = t ~ w) or (k x t = w) or (k = w ~ t); at least one or more of each said (P)t, (P)k, wo oonr~6 rcrnrs~ni8~

and (M~" is an oligonuc~ide of su~cieat length to stably interact independently with a target sequence (the target can be an RNA, DNA or RNA1DNA mixed polymers); D
and E are oligonuclootides which arc greater than or equal to 4 and preferably less tlma 100 nucleotides in length, more specifically 6, 7, 8, 9,10,11,12, 15, 20, or 30 and are a of sufficient length to stably interact irrdepa~dcntty with a target sequence (the target can be an RNA, DNA or RNAIDNA mixai polymers); The D and E oligonucleotides may be symmetric (D = E in krrgth) or asymmernic (D ~ E in length); each B and B' indep~atlY ~praents a cap stNCtun which may independently be present or absent;
and represents a cham~el linkage (e.g. a phosphate ester linkage, amide linkage, i o phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosplwnate or others known in the art).
Formula IV:
A B
(N ~ N')o (S) i s where, N represents a ribonucleotlde which may be the saau or diffd~cnt;
N' is a nucleotide complenae~ty to N; ~ indicates hydrogen bond formation between two ' adjacent ribonucleo$des; o is an integer greater than or equal to 3 and less than or equal to 9, more specifically 4, S, 6, 7, 8 or 9; S, A, and B are oligoribonucleotides which are independently equal to 5 and preferably less than 100 nucleotides in length, more zo specifically 6, 7, 8, 9, 10, 11, 12, 15, 20, or 30; S is an oligonucleatide of sufficient wo oo~m~4c Pcrms~mass ~o length to stably intact independently with a target sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); and ~ a phosplmdicster s Formula V:
(M~
~ ~P~It 1B.
(rF ~ r')h (Z)c d where, each P, N, F, V, Z, and M represents independently a nucleotide which may be same or different; ~ indicates hydrogen bond formation between two adjacent l o nucleotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, S, 6, 7, 8, 9, 10, 11, 12, or 15; k is zero or an integer gt~er than or equal to 3 and preferably less than about 100, more speci5cally 4, 5, 6, ?, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 1~, more specifically 4, S, 6, l s 7, 8, 9, 10, 11, 12, I5, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9,10,11, 12,13, 14,15, or 20; d is an integer greater than or equal to 3 and preferably less than about Z0, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integer gc~er than or equal to 2 and preferably less than about 10, more specifically 2, 3, 4, 5, 6, 7, 8, or 9; c is an integer gt~ter than Zo or equal to 0 and pt~eferabiy less than about 20, more specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; (P), , (Ph, (P)" , (P~,, (V)a and (Z)c are oligonucleotides wo oo~ms ttcrivs9sntttss tt preferably including at least one positron that is not deoxynucleotide (e.g.
2'-H
containing nucleotide); each (P~ ,(P~, (I'~,, (Py, (~d and (2~c may include phosphodiesta, phosphomthioate, plrosphorodithioate, 5'-thiophosphatE, methylplwsphonate linkers and die lika or a combination thereof; k and t may be the s same length (k=t) or different lengths (k ~ t); (lbw is an olig~cleotide sequenex whose inter-nucleotide tinkers msy include phosphodiester, phosphorothioate, S'thiophosphate, methylphospl, or phosphoraditluoate linkers or a combination thereof; t, k, and w may be of the same length (k = t = w) or different length (k ~ t x w) or (k = t ~ w) or (k # t ~ w) or (k ~ w ~ t); at least one or more of each said (P)t, (P~c, ~ o and (M~" is an oligonucleotide of sufficient length to stably interact independently with a target sequence (the target can kx an RNA, DNA or RNA/DNA nuxed polymers);
each B and B' independcatly mpresents a cap swcture wtuch may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphaothioate, phosphorodithioate, 5'-thiophosphate, v a methylphosphonate or others known in the ari). In a preferred embodiment N
andlor N' in (N~N')o, F and/or F' in (F~F'h and/or (~~ , may optionally be able to independently interact with a targ~ sequence.
Formula VI:
(MIw ~ L )t (P)k ~~ ' F 1h iZ)c ( ~t1 B (P''1 (M~

wo oon~34a rcrms~nla6s where, each P, N, F, Z, and M repress indepcndeatly a a>de which may be same or different; ~ i~icates hydrogen bond formation between two adjacent nucleotidca, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 9, 10, 11, 12, s or 1 S; k is uro or an integer greater than or oqual to 3 and preferably less than about 1~, more specifically 4, 5, 6, 7, 8, 9, 10, 1 i, 12, 15, or 20; k1 is zero or an integer greater than or equal to 3 and preferably teas than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, S, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t1 l o is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, I5, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; h is as integer greater than or equal to 2 and preferably less than about 10, more spocifically 2, 3, 4, S, 6, 7, 8, or 9; c is an integer g~ata than or equal to 0 and l s preferably less than about 20, more spocifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,16, or t 8; (P), ,(P~, and (Z)~ is an oligonucleotide preferably including at least one position that is not deoxynucleodde (e.g. 2'-H containing nucleotide); each (P)4 ,(P~, and (Zk may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; k and t may be the zo same length (ht) or different lengths (k # t); k1 and t1 may be the same length (kl~l) or different lengths (k1 ~ t1); (M)" is an oligoaucleotide s«luence whose inter-nucleotide linkers may include phosphadiester, phosphorothioate, 5'thiophosphate, methylphosphonate or phosphorodithioate liakas or a combination thereoF t, k, and w may be of the same length (k = t g w) or different la~gth (k ~ t ~ w) ~ (k = t * w) or (k Zs * t = w) or (k = w x t); t1, k1, and w may be of the same length (k1 = t1=
w) or different length (k1 ~tl xw) or (k1 = t1 ~ w) or (k1 ~ t1 a w) or (k1 = w ~
t1); at least one or more of each said (P)<, (P~, (Py, (P~~,and (M)" is an oligonueleotide of suffcient length to stably intaact independently with a target sequence (the target can WO ti0/17346 PGTNS99/Z1865 l3 be an RNA, DNA or RNAIDNA mixed polymers); each B and B' independently represents a cap structure which may inde~da~dy be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage;
phosphorothioate, phos~orodithioate or others known in the art). In a preferred s embodiment N andlor N' in (N~N')" F andlor F' in (F~F'~, and/or (~~ , may optionally be able to independently interact with a target sequence.
Formula VII:
B' (F ~ F')h Z)c (~ ' I')o B (p_jk ''~ cMn~
where, each P, N, F, V, Z, and M represents independently a nucleotide which l o may be same or different; ~ indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; k is uro or an integer greater than or equal to 3 and preferably less than about 100, more speciFcally 4, S, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is uro or an integer l s greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9,10,11, 12,13, 14,15, or 20; d is an integer greater than or equal to 3 and preferably less than about 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integex greater than or equal to 2 and preferably we oonr346 Pc~rws~ms6s less than about 10, more speafically 2, 3, 4, 5, 6, 7, 8, or 9; c is an integer greater than or equal to 0 and preferably less than about 20, more specifically 1, 2, 3, 4, S, 6, 7, 8, 9, 10, I 1, 12, 16, or 18; (P~ ,(P~, (V), and (Zk are oligonucleoddes preferably including at least one position that is not deoxynucleotide (e.g. 2'-H containing nucleotide); each Ma ~d (Z)c ~Y include phosphodiester, phosphomthioate, phosphorodithioate, 5'-thiophosphate, mathylphosphonate linkers and the like or a combination thereof; k and t may be the same length (k=t} or different lengths (k *t);
(M~" is an oligonucleotide sequence whose inter-nucleotide linkers may include phosphodiester, phosplwrothioate, 5'thiophosphate, methylphosphonate, or ~ o phosphorodithioate linkers or a combination thereof; t, k, and w may be of the same length (k = t = w) or different length (k ~ t ~ w) or (k = t ~ w) or (k a= t =
w) or (k = w $
t); at least one or more of each said (P}t, (P}k, and (Ivl7w is an oligonucleotide of su~cient length to stably interact indepeada~tly with a target sequa~ce (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and 8' independently i s represents a cap structure which may inditpendently be present or absent;
and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonatc or others known in the art). In a preferred embodiment N andlor N' in (N~N')~, F and/or F' in (F~F'), andlor (Z)~ , may optionally be able to independently interact with a target Zo sequence.
Formula VIII:
B B' k D~O
I I
K~T
W

we oonr~s pcrms~ma6s ~s Where, each F, D, O; K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different - length and may include p)un~odiester, phosphorvthioate, S'-thiophospt~ate, _ phosphorodithioate, methylphosphonate linkers and the like or a combination thereof F
s and D ind~artly o~r in combination fornrns RNaxH-activating dotoASin, wherein F
and D are of length greater than or equal to 4 nucleotides if they fornr independent RNaseH domains and are of length grcater-than or equal to 2 nucleotides if they form RNaseH domain in combination; ~ indicates hydrogen bond formation between two adjacent nucleotides within as oligonuclootide; D comprises nucleotide sequence that is i o complementary to the nucleotide sequenct of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O foran 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 base pairs; K and T
form greater than or equal to two base pairs with each other that are contiguous or non-oontigupus, ~ s more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 base pairs;
the K~T and O~D base-paired regions may be contiguous or non-contiguous to each other, K, T, O
and W are of length greater than or equal to 3 nucleotides and ~eferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10,11,12,13, 14,15, or 20; F, D, K, T, W and O togGt>nr are of sufficient lec>$th to stably interact with a target nucleic acid 2o sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B
and B' independently repr~s~tts a cap struchuo which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophoaphate, phosphorodithioate, methylphosphonate or others (mown in the art).
25 FOrmu181X:

o.d _ K~T
Where, each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, s phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; F
and D independently form RNaseH-acdvaring domain, wherein F and D are of length greater than or equal to 4 nucleotides; ~ indicates hydrogen bond formation between two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide l o sequence that is complementary to the nucleotide sequence of T; D and O
form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K
and T form greater than or oqual to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 base pairs; the l s K~T and 4~D base-paired regions may be contiguous or non-contiguous to each other;
K, T, O, and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or ' 20; F, D, K, T, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed Zo polymers); each B and B' independently represents a cap structure which may icukpendently be present or absent; and represents a chemical linkage (e.g. a Phosphate ester linkage, amide linkage, S'-thiophosphate, phosphorothioate, phosphorodithioate, methylphosphonate or otl~s known in the art).
Formula X:
B B' I
F
K~ 'T
W
s Where, each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; F
and D independently form RNaseH-activating domain, wherein F end D are of Length i o greater than or equal to 4 nucleotides; ~ indicates hydrogen bond formation betvveen two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complementazy to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, i s more specifically D and O form 2, 3, 4, 5, 6, ?, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base pairs witb each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 base pairs; the K~T and O~D base-paired regions may ba contiguous or non-contiguous to each other, K, T, O, and W are of length greater than or equal to 3 nucleotides and preferably less 2o than about 100 nucleotides, wore specifically 4, 5, 6, 7, 8, 9, 10, l l, 12, 13, 14, 15, or 20; F, D, K, T, W and O together are of sufficient length to stably interact with a target nucleic said sequence (the target can be an RNA, DNA or RNA/DNA~ mixed wo oon~~ rcrms~nta6s i8 polymers); each B and B' independently represents a cap structure which may independently be present or absent; and represaits a chemical linkage (eg. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'=
thiophospbatc, methylphosphonate or others known in the art).
Formula XI:
B B' I
F

O~D
f I
K~T
W
Where, each F, D, O, K, W and T represents independently as ollgonucleotide whose nucleotide sequence may be same or different, may be of same or different l o length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, mahylphospho~te linkers and the like or a combination thereof; F
and D independently or in combination form ltNaseH-activating domain, wherein F
and D are of length greater thaw or ~ to 4 nucleotides if they form independ~t RNaseH domains and are of length greater than or equal to 2 nucleotides if they form l s RNaseH domain is combin~ion; ~ iadic~tes hydrogen bond formation betw~n two adjacent nucleotides within an oligoaueleotide; D comprises nucleotide sequence that is cmnpleme~ary to the nucleotide Ce of O; K coaadpcises micleotide sequence that is complementary to the nucleotide sequence of T; D and O forni greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more xo specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pails; K
and T form greater than or equal to two base pairs with each other that are contiguous or non-corniguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs;
the K~T and we oonr~a perms~nisss O~D base-paired regions may be contiguous or non-contiguous to each other; K, T, O
and W are of length greater than or oqusl to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; F, D, I~
T, W and O together are of sui~cient ieagth to stably intact with a target nucleic acid sequence (the target can be as RNA, DNA or RNA/DNA mixed polymers); each 8 and B' independently repr~ta a cap structw~e which may indepardartly be presa~t or absent; and its a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the mrt).
i o Formula XII:
B B' D~
w Where, each D, O a~ W mpceseats independently an otigonucleotide whose nucleotide sequence may be sane or diffemat, may be of same or different length and ~ s may include phosphadiester, phosphorothioate, phosphorodithioate, methylphosphonate .. _.. ,,~. ~e ~- ~;a combination thereoF D iadepend~ly forn~s an RNaxH-activating domain of length greater than or equal to 4 nucleotides; ~
indicates hydrogen bond formation between two adjacent nucleotides within an oligonucleotide; D
com~ises nucleotide sequrnee that is complementary to the nuckotide of O;
Zo D and O form greater than or equal to two bane pairs with each other that are contiguous or non-contiguous, more specific~ily D and O form 2, 3, 4, S, 6, 7, 8, 9, 10 or 11 base pairs; O and W are of length greater than or equal to 3 nucleotides and preferably less than about t00 nucleotides, more spxifically 4, 5, 6, 7, 8, 9,10, 11,12, 13,14, IS, or 20; D, W acrd O together are of su~cient length to stably interact with a ~r~~~~
target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' ind~dY its a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. ~' phosphate ester lidcage, amide linkage, phosphorothioate, phosphoroditbioate, s methylphosphonate or others known in the art).
Formula XIII:
B B' O
W
Where, each D, O a~ W represents independently an oligonucleotide whose i o nucleotide sequence may be setae or different, may be of sense or diffarnt length and may inel~e phosphodiester, phosphomthioate, phosphorodithioate, S'-thiophosphate, methylphosphonatc linkers and the like or a combination thereof; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides;
indicates hydrogen bond formation batvveen two adjacent nucleotides within an i s oligonucleotide; D comprises nucleotide sequence that is comptementary to the x...-. . .. ..
nucleotide sequence of OD and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, mare specifically D and O
form 2, 3, 4, S, 6, 7, 8, 9, 10 or 11 base pairs; O and W ere of length greater than or equal to 3 nucleotides and preferably less ttatn about 100 nucleotides, more specifically 4, S, 6, ?, Zo 8, 9,10, 11,12, 13, 14, I S, or 20; D, W and O together are of suflxcient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each 8 aad H' independently represents a cap structiu~e which may independently be present or absent; and re~esents a chemical WO 00V17316 PCTNS99r11$6S

linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphotodithioate, 5'-thiophosphate, methylphosphonate or others known itt the art).
Formula XIV: -B B' I
A
I
o.q K~T
W
Where, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or diffa~ent length and may include phosphodiester, phosphomthioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof;
f o D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; ~ indicates hydrogen bond formation between two adjacent nucleotides within an oligonucieotide; D comprises nucleotide soquence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequencs of T; D and O form greater than or equal to two base pairs f s with each other that are contiguous or non-contiguous, more specifically D
and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T
form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K~T and O~D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length Eo greater than os equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, ?, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, K, T, W and O
tog~her are of sufficient Itngth to stably interact with a target nucleic acid sequence wo oonr~ rcrNS~nts6s n (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap structure which may independentiy be present or absent;
and represents a clxmical linkage (e. g. a phosphate ester linkage, amide linkage;
phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others s known in the art).
Formula XV:
E3 B' I
A
r O~D
I l K~T
w Where, each A, D, O, K, W and T represents independently an oligonucleotide i o whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodicster, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof;
D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; ~ indicates hydrogen band formation between two adjacent nucleotides i s within an oligonucleotide; D comprises nucleotide soquence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide soquence of T; D and O form greater than or equal to two base pairs ' with each other that are contiguous or non-contiguous, more specifically D
and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base Zo pairs with each other that are contiguous or non-contiguous, more specifically K and T
form 2, 3, 4, 5, 6, 7, 8; 9, 10 or 11 base y~irs; the K~T and O~D bast-pairs regions may be contiguous or non-contiguous to each other, A, K, T, O and W are of length we oon~~6 Pernrss9ms6s greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, K, T, W and O
together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA arr RNAlDNA mixed polymers); each B and B' s independently represents a cap structure which may independently be present or absent;
and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophosphate, pho$plmrodithioate, methylphosphonate or others known in the art).
Formula XVI:
io B B' I
A
I
D~
K~T
W
Wlure, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or diffenmt length and may ., include phosphodiester, = phosptmrothioate, 5'-thiophosphate, ~ s phosphoralithioate, methylphosphonate linkers and the like or a combination thereof;
D i~ependently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; ~ indicates hydrogen bond formation between two adjacent n~leotidas within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary Zo to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, S, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base wo oom~ rcrnrs9snis6s is pairs with each other that are contiguous or non-contiguous, more specifically K and T
form 2, 3, 4, 5, 6, ?, 8, 9, 10 or 11 base pairs; the K~T and O~D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of lengds greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, s more specifically 4, 5, 6, ?, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, K, T, W and O
together are of su~cient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap structure which may independently be present or absent;
and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, i o phosphorothioate, phosphorodithioate, methylphosphonate5'-thiophosphate, or others known in the art).
Formula XVII:
B B' A
D~O
I I
K~T
,... ~ . . ..:
~ s Where, each A, D, O, K, W and T repmesents icvdep~dently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof;
D independently forms an RNaseH-activating domain of length greater than or equal to Zo 4 nucleotides; ~ indicates hydrogen bond formation between two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the n~Ieotide sequence of O; K comprises nucleotide sequence that is complementary wo oon~~ rerNS~msss is to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9,10 or 11 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T
s form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K~T and O~D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, S, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, K, T, W and O
together are of suffcient length to stably interact with a target nucleic acid sequence ~ o (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap strucwre which may independently be present or absent;
and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphomthioate, S'-thiophosphate, ptrosphorodithioate, methylphosphonate or others known is the art).
i s Formula XVIII:
B B' A

W
Whore, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and Zo may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides;
indicates hydrogen bond formation between two adjacent nucleotides within an we con»s pcrms~msas oligonucleotide; D compri'es nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O
force 2, 3;
4, 5, 6, 7, 8, 9, 10 or 11 base pairs; A, O and W are of length greater than or equai to 3 s nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, I5, or 20; A, D, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap struchu~e which may independently be present or absent; and represents a chemical i o linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).
Formula XIX:
B B' A
I
O~D
W
~ s , W~~ ~ ~..D, O; and W represents independe~atly an oligonucleotide whose miclootide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides;
Zo indicates hydrogen bond formation between two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complemerrtary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contig~us or non-contiguous, more specifically D and O
form Z, 3, WO OO/I~346 PCTNS99IZ186S

4, 5, 6, 7, 8, 9, 10 or 11 bate pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, W and O together are of sufficient length t6 stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or s RNA/DNA mixed polytners); each B and B' independently represents a cap sttuctw~e which may independently be preseat or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphomthioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).
Formula XX:
io B B' I
I
~
W
Where, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, rs- ~ andlor methytphosphonatie linkers and the like or a combination thereof;
F and D
independently form RNaseH-activating domains of length greater thaw or equal to 4 nucleotides; ~ indicates hydrogen bond formation bet~en two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D arnl O form greater than or equal to two base pairs with zo each other that are contiguous or non-contiguous, more specifically D and O
form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; O and W are of length greater than or equal to 3 nucleotides and preferably less thaw about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; F, D, W and O together are of su~cient length to WO 00/1?346 PCTNS99/Z1863 stably interact with a target mxleic acid aequenoe (tbe target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cep strnchu~e which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5'-s thiophosphate, phosphorodithioate, methylphoslahonate or others known in the art).
Formula XXI:
B B' F
I
W
Where, each F, D, O, and W represents independently an oligonucleotide whose i o nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothiaate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; F and D
independently or in combination form RNaseH-activating domain, wherein F and D
are of length greater than or equal to 4 nucleotides if they form iadependent RNaseH
_.. . ,.: ~ ~: domains and ate bf lei~th greeter than °o1r equal to 2 nucleotides if they form RNasei~~ ~ ~ .. . . . .
domain in combination; ~ indicates hydrogen bond formation between two adjacent nucleotides within an oligonuclcotide; D comprises nucleotide sequence that is complementary to the nucleotide seqt~mce of O; K comprises nucleotide sequence that is complementary to the nucleotide'oquence of T; D and O form greater than or equal Zo to two base pairs with each other that are contiguous or non-contiguous, more specifically D aad O form 2, 3, 4, 5, 6, 7, 8, 9, ! 0 or ! 1 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, S, 6, 7, 8, 9, 10 or 11 base pairs;
the K~T and WO 00/1736 PCTNS99n1866 O~D base-paired regions may be contiguous or non-contiguous to each other, O
and W
are of length greater their or edual to 3 nucleotides end preferably less than about 100 nucleotides, more spcci6cally 4, 5, b, 7, 8, 9, 10,11, 12, 13, 14, 15, or Z0;
F, D, W and' O together are of suffcieat length to archly interact with a target nucleic acid sequence a (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' indepe~~lY:npres~ts a caP sdveture which may independ~tly be presait or absent;
and represents a chemical linkage (t:g. a phosphate ester linkage, amide linhge, phosphorothioate, prodithioste, 5'-thiophosphate, methylphosphonate or others known in the art).
~ o Formula JO1;II:
B B' I
A
I
W
Wha~e, each A, D, O, and W npres~ts independartly an oligonucleotidc where nucleotide sequence may be same or different, may be of same or different length and . . " ..,:~~: .~ n. ~ ~y ~cludo phosphodiester, p~spi~Orot>xioeta, P~phandithioate, 5'-t):iop>wsPlwte.
methylphosphonste linkers aad the like or a combination thereof; D
independently forms an RNascH~activating domain of length greater than or equal to 4 nucleotides;
indicates hydrogen bond formation betWroen two adjacent nucleotides within an oiigonucleotide; D comprises nucleotide sequence that is complementary to the ac nucleotide sequence of O; D and O form gteata than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O
form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D, W and O together are of sufl'rcient length to stably interact with a target nucleic acid sequence (the target can be ari RNA, DNA or RNAlDNA mixed polymers); each B and B' independently reprase~s a cap structure which may indepaidcntly be presets or absent; and represents a chemical s linkage (c g. a phosphate cafe liniuige, amide linkage, phosph~othioate, phosphorodit6ioate, 5'-thiophosphate, rnethylphosphonate or others known in the art).
Formula XX1II:
B B' I
A
I
W
i o Where, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or diB'ar~et, may be of same or different length and may include phosphodiestcr, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D
independently forms an RNaseH-cctivating doaAain of length greater than or equal to 4 nucleotides;
i a i~~tes hydrogen bond formation between two ~~ adjacent. nucleotides oligonucleotide; D oomnucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O
form 2, 3, 4, 5, 6, 7, 8, 9, I O or 11 base pairs; A, O and W are of length greater than or equal to 3 Zo nucleotides and preferably less than about 100 n~leotides, more specifically 4, 5, 6, 7, 8, 9, 10, I1, 12, 13, I4, 15, or 20; A, D, W and O together are of su~cient length to stably interact with a target nucleic acid sequenx (the target can be an RNA, DNA or 1RNA1DNA mixed polymers); each B and 8' independently its a cap stnxture wo oom~6 rrrius~r~ls~s which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, 5'-thiophosphate,amide linkage, phosphorothioate, phospborodithioate, methylphosphonate or others known in the art).
Formula X3~V:
s B B' I
F
I
.o W
Where, each F, D, O, sad W represents independently an oligomicleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphatc, i o methylphosphonate linkers a~ the like or a combination thereof; F and D
independently or in combination form RNaseH-activating domain, wherein F and D
are of length greater than or equal to 4 nucleotides if they form independent RNaseH
domains and are of length greater than or equal to 2 nucleotides if they form RNaseH
domain is combination; ~ indicates hydrogen bond formation between two adjacent i s nucleotides within as oligonuclootide; D comprises nucleotide sequence that is complementaryr to the nucleotide sequence of O; K comprises nucleotide seqtuace that is complem~tary to the n~leotide sequence of T; D and O form greater than or equal to two base pairs with e~h other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, ?, 8, 9,10 or 11 base pairs; K and T
form greater Zo than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, t0 or 11 base pairs;
the K~T and O~D base-paired regions may be contiguous or non-contiguous to each other; O
and W
are of length greater than or oqual to 3 nucleotides and preferably less than about 100 wo oon~3a6 rcrNS~ms6s nucleotides, more spxifically 4, 5, 6, 7, 8, 9, 10, I 1, 12, 13, 14, 15, or 20; F, D, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap stt~ucdu~e which may independently be present or absent;
s and represents a chemical linkage (e.g. a phosphate ester linkage, 5'-thiophosphate, amide linkage, phosphomthioate, phosphorodithioate, methylphosphonate or others known in the art).
Formula XXV:
B B' I
F
I
D~O
w io Where, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate linkers and the like or a combination thereof; F and D
rs independently form RNaseH-activating domains of length greater than or equal to 4 nucleotides; ~ indicates hydrogen bond formation between two adjacent nucleotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with ' each other that are contiguous or non-contiguous; more specifically D and O
form 2, 3, so 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, I5, or 20; F, D, W and O together are of su~cient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or Wt7 ~1~

RNA/DNA mixed polymers); each B end B' independently represents a cap strucnu~e which may independently be present or absent; and repmsents a chemical linkage (e.g. a phosphate ester linkage, amide linkage. P~P~~~
phosphorodithioate, 5'-thioPlmaphate, medhylphosphonate or others known in the art).
s In a prefrrred embodiment, the invention features an HPH molecule of any of formulae I-III, and V-VII, where the (M)" optionally includes an RNase H-activating region.
By "RNase H-activating region" or "RNase H-activating Region" is meant, a region (generally greater than or equal to 4 nucleotides long, preferably S, 6, 7, 8, 9, 10 i o or 11 nucleotides) of a nucleic acid molecule capable of binding to a target RNA to form, for example, a (M~"~target RNA carnplex that is recognized by cellular ItNase H
enzyme, wlure the RNase H enzyme will then bind to the (M~,~target RNA complex and cleave the target sequance. The RNase H-activating region comprises, phosp~diester, phosphorothioate (preferably four of the nucleotides are i s phosphorothiote substitutions; more spedfically, either 4, 5, 6, 7, 8, 9, 10, or 11 of the nucleotides are phosphomthiote substitutions), phosphorodithioate, 5'-thiophosphate, or methylphosphonate baakbo~ c~nistty or a combination thereof. In addition to one or more backbone chemistries described above, the ItNase H-activating region comprises deoxydbose, arabino, fluorae~rabina or a combination thereof nucleotide Zo . ~~ c~. Those skilled in the art will recognize that ~e foregoing are non-limiting examples and that any combinat'ron of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of RNase H enzyme is within the scope of the definition of the RNase H-activating region and the instant invention.
By "nucleotide" as used he:ein is as rxagn~ed in the art to include natural bases Zs (), and modified bases well known in the art. Such bases are generally located at the 1' p~ition of a ancleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. Tl~se nuckeotides can be unmodified or modified at the sugar, phosphate and/or tease moiety, (also referred to interchangeably as nucleotide WO 60/17346 1'CTNS99IZ1866 analogs, modified nucleotides, non-nat~aal nucleotides, non-standard nucleotides and other, see for example, Unman and MoSwiggen, supra; Fckstein et at., International FC'f Publication No. WO 92/07065; Unman et al., International FCT Publication Na.

7; Uhlmaa dl Poyn~an, su~ara) all of which are hereby incorporated by a reference herein). Facasnpks of modified nucleic acid b~ses are known in the art and have recently been summarized by Limbach et al., 1994, N~leic Acids Res. 22, 2183.
Some of the non limiting examples of base modifications that can be introduced i~o nucleic acid molecules i~lude, inosine, gurine, gyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyk, ~ o aminophenyl, 5-alkylcyddirres (e.g., 5-methylcytidine), 5-alkyluridines (eg., ribothymidine), 5-halouridinc (e.g., 5-bromouridine) or 6-azapyrimidines or 6-~Y1PY~~ (e~8 Yl~dine), propyne, and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra). By "modifiat bases" in this aspect is meant nucleotide bases other thsm adenine, guanine, cytosine and uracil at l' i s position or their equivalents; such bases may be used at any positioa, for example, within the catalytic core of an enzymatic nucleic acid molecule andlor in the substrate-binding regions of the nucleic acid cnolocule.
By "ribanucleotide" is aueant a nucleotide with one of the bases adenine, . . , cytosine, guanine, or uracil joined to the. l' carbon of (3-D-ribo-furanose.
zo By "unmodified nuckeotide" is metuit a nucleotide with one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ~3-D-ribo-furanose.
By "modified nucleotide" is meant a nwclootide that contains a modification in the chemical stzvcture of an unmodified nuckootide base, sugar and/or phosphate.
By "abasic" is meant nucleic acid sugar moieties lacking a base or having other zs clumical groups in place of base at the 1' position.

wo oon~~6 rcrivs~ms6s By "sufficient length" is generally meant an oligonucleotide of greater than or equal to 4 nucleotides, or an equivalent chemical moiety able to bind and interact with a target nucleic acid molecule in solution and/or in a cell under physiological conditions.
By "complementarity" is meant that a nucleic acid can form hydrogen bonds) 5 with another RNA sequence by either traditional Watson-Crick or other non-traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., ribozyme cleavage, antisense or triple helix inhibition. l3eternination of binding free energies for ~ o nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH
Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Pros. Nat. Aced. Sci.
USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785. A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can farm hydrogen bonds (e.g., Watson-Crick base pairing) with a ~ s second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70°h, 80%, 90%, and 100% complementary). "perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
By "stably interact" is meant an interaction of the oligonucleotides with target so nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions). The term shall also mean the interaction of HPH
molecules with the target molecule for a duration, under physiological conditions, in solution or in a cell, sufficient for the HPH molecule to interfere with the function of the target nucleic acid molxule.
as Typically, antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain wo oort~3~ rc~rnrs~ma6s embodiments, an antiaense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop. Thus, the antisense molecule may be complementary to two (8'~
even more) non-contiguous substrate sequences or two (or even more) non-contiguous s sequence portions of an antiasnsa molecule may be complementary to a target sequence or both.
By "nucleic acid molecule" as used herein is meant a molecule comprising nucleotides. The nucleic acid can be composed of modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
E o By "inhibit" it is meant that the activity of target genes or level of mRNAs or equivalent RNAs encoding target genes is reduced below that observed in the absence of the nucleic acid moiecule of the invention. In one embodiment, inhibition with HPH
molecules preferably is below that level observed in the presence of an mismatched nucleic acid molecule that is not able to stably bind to the same sift on the mRNA. In i s another embodiment, inhibition with HPH nucleic acid molecules, is preferably greater than that observed in the presence of for example, an oligonucleotide with scnunbled sequence or with mismatches. In another embodiment, inhibition of target genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absencx. By "inhibit" is also meant, an impediment to 2o normal function of a macromolecule caused by the introduction a foreign substance, such as the HPH molocule.
v, By "target sequence" or "target nucleic acid molecule" is meant, a gene or partial sequence thereof, and those elements necessary for its expression, regulation, or its transcription or replication product or intermediates or portions thereof, including zs DNA, RNA or protein.. Non-limiting examples of target sequence include c-raf mRNA, hepatitis C RNA, vascular endothelial growth factor receptor (e.g.. flt- and ICDR), ras wo oonr~s pcrms~nisss RNA, and the like.
By "gene" it is meant a nucleic acid that encodes an RNA.
By "antisense" is meant a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., s 1993 Nature 365, 566 interactions and alters the activity of the target RNA
(for a review see Stein and Cheng,1993 Sciaece 261, 1004).
By "equivalent" RNA to target genes is meant to include those naturally . .. .
occurring RNA molecules having homology (partial or complete) to genes or encoding for proteins with similar function as genes in various animals, including human, rodent, ~ o primate, rabbit and pig. The equivalent RNA sequence also includes in addition to the coding region, regions such as S'-untraoslated region, 3'-untranslated region, introns, intron-exon junction and the like.
By "related" is meant that the inhibition of target gene RNAs and thus reduction in the respective levels of protein activity will relieve to some extent the symptoms of l s the disease or condition.
By "cap structure" is meant chemical modifications which have been ~~~,~e. n~ of the oligonucleotide (e.g., B and B' in formulae above).
These terminal modifications protect the nucleic acid molecule from exonucleese degradation, and may help in delivery and/or localization within a cell.
zo In another preferred embodiment, (P~, (P)n (N'N')~, (F~F'),, (V)d, (Z)~
(P~,. (P)«, (M)", (~" (~~, D, K, T, W andlor E, independently include modifications selected from a group comprising 2'-O-alkyl (e.g. 2'-O-allyl; Sproat et al., supra) sometimes referred to as RNA modifications; 2'-O alkylthioalkyl (e.g. 2'-O-methylthiomcthyl;
Karpeisky et al., 1998, Nucleosides & Nucleotides 16, 955-958); L-nucleotides zs (Tazawa et al., 1970, Biochemistry 3499; Ashley, 1992, J. Am. Chem. Soc.
114, 9731;

wo oonr~6 pcrms9sma6s Klubmenn et a1.,1996, Nature Biotech 14,1112); 2'-C-alkyl (Beigelman et a1.,1995, J.
Biol. Chem. 270, 25702); 1-5-Anhydmhexitol; 2,6-diaminopurine (Strobel et al., 1994, Biochem. 33, 13824-13835); 2'-(N atanyl) amino-2'-deoxynucleotide; 2'-(N bets=
alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino (Karpeisky et al., 1995, Tetrahedron Lett. 39, 1131); 2'-deoxy-2'-(N histidyl) amino; 5-methyl (Strobel, supra);
2'-(N b-carboxamidine-beta-alanyl) amino; 2'-deoxy-2'-(N-beta-alanyl) (Matulic-Adamic et al., 1995, Bioorg. & Med. Chem. Lett. 5,2721-2724); xylofiuanosyl (Rosemeyer et a1, 1991, Helvettca Chem. Acts 74, 748; Seela et al., 1994, Helvetica Ci~m. Acts, 77, 883; Seela et al., 1996, Helvetica Chem. Acts, 79, 1451).
i o In yet another preferred embodiment, B' is selected from a group comprising inverted abasic residue,. 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclie nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha nucleotides; modified base nucleotide; phosphorodithioate linkage;
threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-i 5 dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypemyl nucleotide, 3'-3'-inverted nucleotick moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate;
hexylphosphate;
aminohexyl phosphate; 3'-phosphate, phosphorothioate (preferably three of the terminal nucleotides are phosphomthiote substitutions; more specifcally, either 1, 2, 3, 4 or 5 of Zo the 'terminal nucleotides are phosphorothiote substitutions); ~
phosphoiodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Beigelman et al., International PCT publication No. WO 97126270, incorporated by reference herein).
In yet another preferred embodiment, B is selected from a group comprising, 4',5'-methylene nucleotide; I-(beta-D-erythrofutanosyl) n~leotide; 4'-thio nucleotide, Zs carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; I,2-aminododecyl phosphate;
hydroxypropyl phosphate; 1,5-anhydmhexitot nucleotide; L-nucleotide; alpha-we oon~as rcnus99ma6s nucleotide; modified base nucleotide; phosphorodithioate; three-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, S'-5'-inverted nucleotide moeity; 5'-5'-inverted abasie moeity; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino;
bridging and/or non-bridging 5'-phosphaa~nidate, phosphorothioate (preferably three of the terminal nucleotides are phosphomthiote substitutions; more specifically, either l, 2, 3, 4 or 5 of the terminal nucleotides are phosphorothiote substitutions); and/or phospharodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925;
i o ~ incorporated by reference herein).
An "alkyl" group refers t0 a sawratod aliphatic hydrocarbon, including straight chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl gmup may be substituted or unsubstituted. When substituted i s the substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or N(CH3)2, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 . . ;.to 12 carbons. ~ More ,preferably it is a lower alkenyl of from I to 7 carbons, more zo preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted.
When substituted the substituted groups) is preferably, hydroxyl, cyano, alkoxy, ~, =S, N02, halogen, N(CH3)2, amino, or SH. The term "alkyl° also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
Zs Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably I to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted groups) is preferably, wU 00/19346 PCTlUS99/21865 hydroxyl, cyano, alkoxy, ~, =S, NOZ or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocycl~
aryl, amide and ester groups. An "aryl" group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, s heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl gmups are halogen, trihalomtthyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group refers to an.alkyl group (as described above) oavalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring ~ o are all carbon atoms. The carbon atams are optionally substituted.
Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
l s An "amide" refers to an -C(OrNH-R, where R is either alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to an -C(O~OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
In a preferred embodiment, the HPH molecules including the molecules described .. ~ , ,. ., . . .,..
in formulae I-XXV are capable of binding to the target nucleic acid molecules in a Zo sequence-specific manner. The stable interaction between the HPH molecule and the target molecules interferes with the normal function of the target molecule.
Such interaction, for example, tray cause inhibition of the function of the target molecule, such as transcription, translation, and replication: The HPH molecules of the invention interact and interfere with the target molecule In vitro or in viva in a bacterial cell, is microbial system, plant system, or mammalian system to modulate the function of the target molecule in such biological systems. In a prefernd embodiment, the HPH
molecules of the instant invention are used to inhibit target-gene expression in a wo Qa/17346 PCT/US99/21865 biological system, more specifically in a cell, tissue, organ, and animal.
In a preferred embodiment, the HPH nucleic acid molecules including the molecules of formulae I-III and V-XXV comprise at Least one phosphate backbone modification, where such a modification is phosphorothioate (preferably four of the s nucleotides have phosphomthiote substitutions; more specifically, either 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 19, 21, 23 or 25 of the nucleotides have phosphorothiote substitutions), phosphomdithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.
In a preferred embodiment, the HPH nucleic acid molecules including the l o molecules of formulae I-XXV are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex viva, or in viva through injection, infusion pump cx stem, with or without their incorporation in biopolymers.
l s In another aspect of the invention, the HPH nucleic acid molecules described in formulae IV are expressed from transcription units inserted into DNA or RNA
vectors.
The recombinant vectors are preferably DNA plasmids or viral vectors. HPH
molecule . . . expressing viral vactors~ could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Preferably, the recombinant Zo vectors capable of expressing the HPH molecules axe delivered as described above, and persist in target cells. Alternatively, viral vectors may be used that provide for transient expression of HPH nucleic acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecules bind to target mRNA. Delivery of nucleic acid molecules expressing vectors could be systemic, such za as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510). In another aspect of the invention, nucleic acid molecules that bind target molecules and inhibit cell proliferation are e~cpr~od fmm transcription units inserted into DNA, RNA, or viral vectors.
Preferably, the recombinant vectors capable of expressing the HPH molecules are locally delivered as described above, end transiently persist in smooth muscle cells.
However, other mammalian cell vectors that direct the acpnssion of RNA may be use for this purpose.
By "ph~otype" is meant, the entire physical, biochemical, and physiological l o makeup of an organism at determined both genetically or environmentally and any one or any group of such traits.
In a preferred embodiment, the 5' andlor 3' portions of the hairpin region of the HPH molecule is independently complementary to the target sequ~eace.
Specifically, N
andlor N' portion of the (N~N')~ in formulae I-VII is independently complementary to l s the target sequence.
In a preferred embodiment, tlu 5' andlor 3' portions of the hairpin region of the HPH molecule is independently complementary to the target sequence.
Specifically, N
. ., ., ..,... , . . . ~dtor.N'.portion or ther(F~F'h in formulae..d-VII
is.independently complementary to . . _ . ...
the target sequence.
zo In a preferred embodiment, the invention features a method of modulating the function of a target sequ~ce including the steps of contacting the target sequence with the HPH molecules, including the molecules of formulae I-X7N, under conditions suitable for the modulation of the function of the target sequence. Such modulation can take phux in vitro or in vivo, in microbial, plmo~, ar mammalian systems where the is modulation of function may include inhibition of gene expression, modification of wo oon~3ls rcrros~nisss cellular function, change in the organism's phenotype, inhibition of replication of a virus and/or viral RNA, inhibition of motility, migration of a cell and others.
By "patient" is meant as organism that is a donor or recipient of explanted cells or the cells th~rselves. "Patient" also refea~s to an organism to which enzymatic nucleic s acid molecules can be administered. Preferably, a patient is a mammal or mammalian calls. More preferably, a patient is a human or human cells.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
In another aspect, the nucleic acid molecule of the present invention is i o administered individually or in combination or in conjunction with other drugs, and can be used to treat diseases ~ co~itions. For example, to treat a disease or condition associated with cancer, the patient may be treated, or other appropriate cells may be tn~d, as is evident to those skilled in the art.
By "comprising" is meant including, but not limited to, whatever follows the i s word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or taandatory, but that other elements are optional and may or may not be Wit. By. "~'°o$ o~ is meant including, and limited to, whatever ~._ : ....... ,.. :..., .. _ ..
follows the phrase "consisting of . Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elegy may be so By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified is the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are roquitrd or mandatory, but that other elennents are optional and may or may not be present depending upon whether or zs not they affect the activity or action of the listed elements.

we oon~~ rcrms~ms6s 4a Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The drawings will first briefly be described.
Figure 1 is a schmaatic rep~e~ntation of the binding of the hairpin hybridi~er (HPH) molecule to a target RNA. During binding, both the 5' and 3' sequences of the hairpin region may be non-complementary to the target sequence. Alternatively, either the 5' or 3' sequence may be complementary to the target RNA molecule i o independently.
Figure 2A displays the hairpin structure of the unbound HPH nucleic acid molecule including a 4 base pair stern and an internal 9-nucleotide DNA
sequence. The figure further displays the structure of the nucleic acid molecule before and after binding to RNA. This molecule's 5' and 3' sequences form the hairpin structure but do i 5 not base pair with the target RNA molecule. Figure 2B displays the hairpin structure of the uabound nucleic acid molecule also including a 4-base-pair stem and an internal 9-nucleotide DNA. sequence. This molecule's 5' and 3' sequence forms the hairpin structure. In certain embodiments, the 5' andlor 3' sequence is capable of binding to the target RNA molecule independently.
2o Figure 3 displays non-limiting stn~ctures of the HPH molecules that are within the scope of the present invention. In Figure 3A, (1) represents a circular nucleic acid molecule with an internal base-paired hairpin stem structure, each loop within the ', molecule comprises an RNase H-activating Region and a Non-RNase H-activating Region aad is capable of binding to a Target Sequence; (2) represents a molecule 25 COmpIlSlng an RNase H-activating Region and a Non-RNase H-activating Region WO 00/1736 PGT/US99/Z186d capable of interacting with the Target Sequence and includes an internal base-paired hairpin stem region and additional nucleotide sequences at the 5' and 3' ends of the hairpin structure which are of equal or dissimilar lengths that may optionally bind to Target Sequences; (3) represarts a molecule comprises an RNase H-activating Region s and a Non-RNase H-activating Region capable of interacting with the Target Sequence and an internal base-paired hairpin stem region and additional nucleotide sequence at the 5' end of the hairpin stem structure which may optionally bind to Target Sequence;
(4) represents a molecule comprising an RNase H-activating Region and a Non-RNase H-activating Region capable of intenzcting with the Target Sequence and an internal i o base-paired hairpin stem region and additional nucleotide sequences at the 3' end of the hairpin structure that may optionally bind to target sequence; (5) represents a molecule comprises an RNase H-activating Region and a Non-RNase H-activating Region capable of interacting with the Target Sequence and an intennal hairpin stem region and additional nucleotide sequences at the 5' and 3' ends of the hairpin structure which are i s asymmetric in length and which may optionally bind to Target Sequences; (~
represents a discontinuous circular nucleic acid molecule comprising an RNase H-activating Region and a Non-RNase H-activating Region capable of interacting with the Target Sequence and an internal hairpin stem structure, each loop at the 3' and the 5' ends of the hairpin region is indepe~ently capable of binding to a target sequence. In eo Figare 3B, (1) repi~eserits a HPH nucleic acid molecule structure, where~the RNase 1~'-activating Region is at the 5' end of the molecule and a portion of the RNase H-activating Region forms a hairpin stem structure with a portion of the 3' region of the HPH molecule. Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target 25 sequence in a sequence-specific manner, (2) and (5) represents a HPH
nucleic acid molecule structure, where a portion of the RNase H-activating Region forms a hairpin stem structure with a portion of the non-RNaxH-activating region of the HPH
molecule. Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a soquenco-specific manner, (3) and (6) reprint a HPH nucleic acid molecule sr<ucdue, where the a portion of the RNase H-activating Region and a poartion of the Non-RNase H-activating region forms a hairpin stem structure with a portion of s the Non-RNase H-activating region located in a diffet~t part of the HPH
molxule.
Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner, (~) npraents a HPH nucleic acid motaaile structure, where the RNax H-activating Region is at the 3' end of the molecule and a portion of l o the RNase H-activating Region forms a hairpin stem structure with a portion of the 5' region of the HPH molecule. Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target soquence in a sequence-specific manner. In Figure 3C, {1) and (3) a HPH nucleic acid m~ecule ~ a portion of the RNase H
i s activating Region forms a hairpin stem structure with a portion of the non-RNaseH
activating region of the HPH molecule. Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequenco-spxific manner; (2) and (4) represents a HPH nucleic acid molecule sin~cdu~e, where the a portion of the RNase H
zo activating Region aril a portion of the Non-RNase H-activating region form a hairpin stem structure with a portion of the Non-RNase H-activating region located in a different part of the HPH molecule. Both t~ RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target s~equeace in a sequence-specific manner.
25 Figure 4 displays a gtoph demonstrating the effect of a 3lmer HPH nucleic acid molecule of the present invention on redu«ng c-raf mRNA levels in PC-3 cells compared to untreated and mismatch oa~ols. Tha cells were treed with nucleic acid molecules for 1, 3, or 5 days and then harvested to quantify the o-raf RNA.

wo oom346 ~c~rnrs~ms6s Figure 5 displays a graph demonstrating the effect of a 33mer nucleic acid molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to untreated and mismatch coatrals. The cells were treated with the HPH
nucleic acid molecules for 1, 3, or 5 days and then harvested to quantify the c-raf RNA.
s Figure 6 displays a graph demonstrating the effect of a 35mer HPH nucleic acid molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to untreated and mismatch controls. The cells were treated with nucleic acid molecules for I, 3, or S days and then harvested to quantify the c-raf RNA.
Figure 7 displays a graph demonstrating the effect of a 31 roes HPH nucleic said t o molecule of the prrsent invention on reducing c-raf mRNA levels in PC-3 cells compared to a mismatch control.
Figure 8 displays a graph demonstrating the effect of a 31 roes HPH linear antisense molecule on crducing c-raf mRNA levels in PC-3 cells compared to a mismatch control.
t s Figure 9 displays the HPH nucleic acid molecule-based specific inhibition of c-raf RNA levels in PC-3 cells and the e$ect of l, 2 and 4 base mismatches on this inhibition.
Figure 10 displays several non-limiting examples of psuedolcnot hairpin hybridizes molecules. Figure 10A is a psuedoknot hairpin hybridizes molecule Zo comprised of 2 hairpin structures, and a target binding sequence located in closer proximity to the 5' end of the nucleic acid molecule compass=d to the 3' end.
Figure lOB
is a psuedoknot hairpin hybridizes molecule comprised of 2 hairpin structures, and a target binding sequence located in closer proximity to the 3' end of the nucleic acid molecule compared to the 5' end. Figure IOC is a psuedoknot hairpin hybridizes 2s molecule comprised of 2 hairpin struchurs, and two target binding sequences. Figure lOD is a psuedolcnot hairpin hybridizes molecule comprised of 2 hairpin structures, a target binding sequence located in closes proximity to the 3' end of the nucleic acid molecule compared to the S' end, and additional nucleotide sequences attached at the 5' wo oom3~ pcrms~a~8as and 3' ends of the hairpin hybridi2er mol~ule. T~Se additional sequences may be of equal or unequal length. Figure 10E is a psuedoknot hairpin hybridizes molecule comprised of 2 hairpin stxuetures, 2 target binding sequences, and additional nucleotide sequences attach at the S' and 3' ends of the hairpin hybridizes molecule.
These a additional sequences may be of equal or rmequal length. Figure lOF is a psuedoknot hairpin hybridiur molecule compr~d of 2 hairpin swctures, a target binding seqc~ence located in closer proximity to the 5' end of the nuclrac acid molecule compared to the 3' end, and an additional nucleotide sequa~ce attached at the 5' of the hairpin hybridizes molecule. Figure lOG is a psuedoknot hairpin hybridizes molecule i o comprised of 2 hairpin structures, a target binding sequence located in closer proximity to the 5' end of the nucleic acid molecule compared to the 3' ead, and additional nucleotide sequences attached at the 5' and 3' ends of the hairpin hybridizes molcxule.
These additional sequences may be of equal or unequal length.
Figure 11 displays a graph demonstrating the effext of HPH nucleic acid mo<ecule i s of various configurations of the pit invemion on reducing o-raf mRNA
levels in PC-3 cells compared to controls.
Figure 12 displays a graph demonstrating the effect of a HPH nucleic acid molecule of the varying sizes and configurations of the present invention on reducing IMPDH II mRNA lavels in PC-3 cells compared to untreated and mismatch controls.
Eo Figure 13 displays a graph demonstrating the effect of a HPH nucleic acid molecule of the varying sins and configurations of the present invention on enduring IMPDH II mRNA levels in PC-3 at an oligonucieotide conc~ation of 100 nM.
~Vlxhanism of actiyg of 'Ihe HPH Nuclenc Acid Molecules of the Im~entio~n as Antisense molecules known in the art are usually RNA or DNA
oligonucleotides and primarily function by specifically bindurg to complementary (matching) sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BivPltarm, 20-33).

we oon~~s Pcrius99msss The oligonuclootide binds to target RNA by Wat9on Crick base-pairing and blocs gene exon by prevetttiog ribosomal translation of the bound sequences by either steric blocking or RNase H-mod~ieted d~agradation of target RNA. Antisense raoleoules may also alter protein synthesis by inta~ing with RNA processing or trm~sport from s the nucleus into the cytoplasm (Mukhopadhyay dt Roth, 1996, Crit. Rev fin Oncogerresls 7,151-190).
In addition, binding of single stiaaded DNA to RNA may result in nucl~se degradation of the heteroduplex (Wu-Poaig, supra; Crooke, supra). To date, tl~
only bac3cbone-modified DNA chemistry which will apt as substrates for RNase H are i o phoaphorothioates and phosphorodithioates. Recently it has been reported that 2'-arabino and 2'-fluoro arabiao- containing oligos can also activate RNase H
activity.
A number of antisense molecules have been described that utilise novel configurations of chemically modified nucleotides, secondary structure, and/or RNase H substrate domains (Woolf et al., International PCT Publication No. WO
98/13526;
i s Thompson et al., USSN 60/0$2,404 which was filed on April 20, 1998 both of which are incorporated in their entirety by referaxx herein).
The antisense molecules described in the art are essentially single-stranded linear oligonuclootides which ~e known to tolerate a numbex of mismatches and still form stable hybrids with a target sequenx raising the coaoem of safety and toxicity in Zo organisms. While these molecules are fitnctional, for certain epp<ic~tions, including pharmaceutical compositions, gcaatcr spociftcity, lower toxicity sad higher stability is desirable.
The specificity of oligonucleotides d~ribod above may be inched by using tl~ HPH nucleic acid molecule of the prescrtt invention which form internal hairpin Zs structure with hydrogen bond interactions. Partial complementarily of these HPH
oligonucleottdes with target sequences do not allow for e~cient opening of the intenaal hairpin str~ture of the HPH oligonucleotide resulting in a competition betwun a WO OQlt7316 P4TNS99/11863 hairpin structure and binding to target a sequence. Therefore, hybridization interaction betwoen HPH and target sequences with one or more mismatched sequences, occurs ine~ciently, thereby malting them iae~cient inhibitors of gene expression (see fig.
9).Tyagi & ICramer, 1996, Nat BfotecJirrol 14,303-308; Tyagi & ICramer, 1998, Nat a Biotechnol 16, f59-363 have at~Wn that oligonucleotides stub as molecular beacons which have a 10 base pair or more int~aa! hairpin stems are capable of binding to a target sequence in a highly seqtrerue specific manner in solution. The specific interaction of a hairpin DNA with target RNA was also demonstrated in cells (ICostrikis. et a~, 1998 , .Science 2?9, 1228-1229) where the hairpin DNA was used to ~ o detoct the press of bFGF RNA, thex oligonuclootidas howrver were ~t used to inhibit gene expression.
In addition to the in~xd~d specificity, the intramolecu>ar bonding of the hairpin hybridizer moloeules can result in increased stability. Hairpin sequences locabod at the respective cads of the oligontrcleotide may increase the stability of these t s tits because the lack of turpaitcd free nucleotides redtuxs the potential for degradation by exonuclaises. find stabilization is cturently conferred by clxmieal modifications (p>msp~rothioate linkage etc.) which may itself specificity, and possibly increase cytotoxicity. The inctaased stability of hairpin-end vector-based ribozyme constructs has already been demonstrated (fhompson et al., 1995, Nucleic ac Acids Research 23, 2259-2268).
T6e effectiveness of these HfH molecules may be enhanced by the addition of nucleotides which act as substrates for RNase H within the molecule. However, binding of DNA to RNA is not as thermadynamically favorable as an RNA to RNA
interaction (Alttnann et al.,1996, Chimla 50,168-1?6). Therefore a molecule with both Zs RNA and DNA nucleotides may be able to bind efficiently as well as promote degradation of the RNA molxule by RNase H. Inoe & Ohtsuka, 198?, Nucleic Acids Research I 1 S, 6131, first proposed an oligonucleatide with a central region consisting of oiigodeoxyaucleotides flankred by 2'-O-methyl modified nucleotide regions.
The wo oonr~s pcrius~nm s~
region of otigodeoxynucleotides in such a chimeric molecule is rocogniud by RNase H
when bound to RNA; and facilitates cleavage of target RNA by RNase H. (Inoe 8t Ohtsuka, 1987, FEBS Lett. 215, 327; Shibnhara 8c Morisava, 1987, Nucleic Aeict~
Res. 15, 4403). These chimeric oligoaucleotides were proposed to interact with target s RNA more stably than an all DNA oligonucleotide. Alternatively, the nucleic acid ~kcule may fimcrian by binding to tlu: target molecule that results in staric hindrance for ribosomal translation. A number of chemical modi5cations may be utilized with this strategy including insertion of 2'-l~.methyl modifi~ion at every nucleotide in tl~
molecule.
i o One of the most stadicd and util~d chemical alterations in oligoaucleotides has been backbone modifications such as pha~pbmothioates, phoaphorodithioat~, and 5'thio~. Phosphomthioate oligonucleotides are nucleic acid molecules whose phosphodiester linkage has been modi$ed by substituting a sulfur atom in place of an oxygen atom. In addition to increased nuclease resistance, phosphorothioate, i a phosphorodithioate, and 5'thiophosphates oligonueleotides are substrates for ribonuclease H (RNase H) (Modria, supra; Crooke et al., 1995, Bixhem. J. 3112, 608). RNase H is an endonuclease which catalyzes the degradation of RNA in an RNA-DNA hetemduplex (Hostomsky et al., 1993 in Nucleases, Lien et al., ads., Cold Spring Harbor Laboratory Press, NY, 341-376). RNA/DNA heteroduplaces, called Zo Okazaki fragments, ante formed naturally during DNA replication. Therefore, the nornsal function of RNase H is to degrade the RNA portion of the hetaoduplex to complete DNA replication. in experiments with E. Colt RNase H, the phosphorothioate oligonucleotide activated the enzyme more efficiently (2-s fold) compared to a standard phosphodiester containing oligonucleotide (Crooke,1995, supra).

wo oon» rcrius99ntaas s2 ~myesis of Nucleic acid Molecules Synthesis of nucleic acids r than 100 nucleoti~s in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
In this iuve~ion, small nucleic acid motifs ("small refers to nucleic acid motifs no more than s 100 nucleotides in length, preferably no more t3~an 80 nucleotides in length, and most preferably no more than 40 nucleotides in length; e.g., HPI-I nucleic acid molecules) are used for exogenous delivery. The simple stnxxure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure. The oligodeoxyribonucleotides molecules of the instant invention were chemically l o synthesized using standard protocols as described in Caruthers et al., 1992, Methoals in Emymology 211,3-19, which is incorporated herein by reference.
The method of synthesis used for normal RNA follows the pnxedure as described in Unman et al., 1987 .J. Am. Ci~em. Soc., 10'9, 7845; Scaringe et at., 1990 Nucleic Acids Res., 18, 5433; and Wincott et al., 1995 Nucleic Aclds Res. 23, 26?7-l s 2684; Wincott et al., 1997, Methoals Mol. Bio., 74, 59) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end and can be readily used to synthesize 2'-D-alkyl-containing oligonucleotides. In a non-limiting example, small-scale synthesis was conducted on a 394 Applied Hiosystems, Inc. synthesizer using a modified 2.5 l,unol Zo scale protocol with a 5 min. coupling step for alkylsilyl protected nucleotides and 2.5 min. coupling step for 2'-D-methylated nucleotides. Table I outlines the amounts, and the contact tunes, of the reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2 Wnol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
A IS-is fold excess (31 NL of 0.1 M = 3.1 Na~ol) of phospltoranaidite and a 38.7-fold excess of S-ethyl tebrazvle (31 pI, of 0.25 M = 7.75 pmol) relative to polymer-bound 5'-hydroxyl was used in each coupling cycle. Average coupling yields on the 394 Applied WO 00/19346 1'CfNS99121866 Bioa~ystems, Inc, synthesis, determined by colorimetric quatititation of the trityl fractions, were 97.5~99~/0. Other oligonucleotide synthesis reagems used with the 394 Applied Biosysten~s, Inc. synt>>esiza included detritylation solution with 3%
TCA-ta methylene chloride (ABI); capping was performed with 16% N methyl imida~le in s THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABn; oxidation solution was 16.9 mM IZ, 49 mM pyridine, 9% water in THF (PERSEPTIVE'i'"~.
Burdick 8t Jackson Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acctonitrile) was made up, from the solid obtained firm American Inonal Chemical, Inc.
i o Deprotection of the the oligonuclaotides of the instant invention was performed using either a two-pot or one-pot protocol. For the two-pot protocol, the polymtr-bound trityhn oligoribonueleotide was transferred to a 4 mL glass screw top vial and suspended in a solution of 40~/° aq. methylamine (1 mL) at 65 °C
for 10 min. After - cooling to -20 °C, the supernatant was removed from the polymer support. The ~ s support was washed three times with 1.0 mL of Ett?H:MeCN:H20/3:1:1, vortexed and the supei~tant was then added to the fast supernatant. The combined supen~atants, containing the oligoribonucleotide, were dried to a white powder. The base deprotected oligoribonucleotide was resuspended in anhydrous TEA/HFINMP solution (300 ~I, of a solution. of 1.5_ tnL N-metbylpyrrolidinone, 750 uL TEA and 1 mL TEA~3HF to zo provide a I .4 M HF con~tration) and heated to 65 °C. After 1.5 b, the oligomer was qvenchai with 1.5 M NH4HC03.
Alternatively, for the one-pot protocol, the polymer bound trityl-on ' oligoa'bonucleotide was traruferKd to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic ~thyl~nine/DMS0:1/1 (0.8 mL) at 65 °C for 15 min. The ', as vial was brought to room tttnperature. TEA~3HF (0.1 mL) was added and the vial was heated at 65 °C for 15 min. The sample was cooled at -20 °C and then quenched with 1.5 M NH~HC03.

wo oonr~s rcrNS99msss s~
For puri8cadon of the trityl-on oligomers, the quenched NH.~HC03 solution was loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA
was detritylated with 0.5% TFA for 13 rain. The cartridge was then washed again with s water, salt exchanged with 1 M NaCI and waslud with water again. The oligonucleotide was then ehrted with 30~/o a~cetonitrile. Alternatively or in addition to the methods described herein, oligonuclootides of the instaat inventions can be purified by other methods known in the art, for example, see Sprout et aL, 1999, Nucleic Acids Res., 27, 1950).
i o The average stepwise coupling yields were >98% (Wincott et al., 1995 Nucleic Acids Rep 23, 2677-2684). Thox of orditutry skill in the art will racogiiize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limitai to 96-well format, all that is important is the ratio of chemicals used in the reaction.
a s Alternatively, the nucleic acid molecules of the pint invention can be synthesized separately and joined together post synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT
publication No. WO 93/23569; Shabarova et al., 1991, Nucleic Acfds Research 19, 4247;
Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997 Bfocon,Jugare Chem.
2o 8, 204).
Adtainistration of Nucleic Ac;g~~,~ecules Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bfo., 2, 139; and Delivery Strategies for Antfsertse Oligonucleotide 77reraperafcs, ed. Akhtar,1995 which are both incorpon~ed herein by reference.
Sullivan et a5 a1, PCT WO 94/02s95, fia~dux describes the general m~Ods for delivery of enzymatic RNA molecules. These protocols may be utilized for the delivery of virtually any nucleic WO 00/1346 PCTlUS99t11865 ss acid molecule. Nucleic acid trmleettles may be administered to calls by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophotasis, or by inc~pocahon into other vehicles, such as hydrogeis, cyclodextrins, biodegradable nanocapsules, and bioadhesive microsphens. For some indications, nucleic acid molecWiea may be directly deliveccd ex vivo to sells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid/vehicle combination is locally detiv~ed by dit~ect injection or by use of a catheter, infusion pomp or stmt. Other mutes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, i o intrapaitoneal and/or intrathecal delivery. More detailed descriptions of nucleic-acid delivery and administration are provided in Sullivan et al., supra and Dn~per et al., PGT
W093/23569 which are incorporatod by refa~x herein.
The molecules of the instant iavetaron can be used as pharmaceutical agents.
Pharmaceutical agents prevent, inhabit the occarrence, or treat (alleviate a symptom to l s some extent, preferably all of the symptoms) of a disease state in a patient.
The negatively charged polynucleotides of the invention can be administered (eg., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilisers, buffers, and the I~Ce, to form a pharmaceutical composition.
When it is desired ' to use a liposome delivery mechanism, standard protocols for Zo formation of liposomes can be folloWCd. The compositions of the presait invention may also be formulated and used as tablets, capsules or elixirs for oral administration;
suppositories for eater administration; sterile solutions; suspensions for injectabte administration; and the like.
., The present invention also includes pharmaceutically acceptable formulations of z a the compounds described. These formulations include salts of the above compounds, we oam34s rcrnls~m ess as e.g., acid addition sans, for exannple, salts of hydrochloric, hydrobromic, acetic acid, and benmene sulfonic acid.
A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a s cell or patient, preferably a human. Suitable forms, in part, depend upon the use or tire route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble.
Other l o factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or fen from exertiag its effect.
By "systemic administration" is meant in vivo systemic absorption or accumulation of drugs in the blood stn~m followed by destribution throughout the entire body. Administration routes which lead to systemic absorption include, without ~ s limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. Each of these administration routes expose the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue. The rate of entry of a drug into the circulation has been shown to be a function of molecular weight or siu. The use of a liposome or other drug carrier comprising the compounds of the eo instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES). A liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhaaced delivery of the drug to target cells by taking advantage of the specificity of macrophage Za and lymphocyte immune recognition of abnormal cells, such as canxr cells.

we oonr34s PcrNS~ais6s s~
The invention also features the ~e of the composition comprising surface-modified liposomes containing poly (ethyle;ne glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomos). These formulations offer an method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists s opsoni?ation and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasie et al. Chem. Rev 1995, 95, 2b01 2627; Ishiwata et al., Chenr.
Pharm. Bulb 1995, 43, 1005-1011). Such lipoaomes have been shown to accumulate selectively in tumors, presumably by exhavasation act capture in the neovascularized-i o target tissues (Lasic et al., Science 1995, 267, 12?5-1276; Oku et a1.,1995, Biochim.
Biophys. Acta, 1238, 86-90). The long-circulating liposomcs enhance the pbaimacokinetics and pharrnacodynamies of DNA and RNA, particularly compared to conventional cationic liposomas which arc known to accumulate in tissues of the MPS
(Liu et al., J Biol. Chern. 1995, 42, 24864-24870; Choi et al., International PCT
i s Publication No. WO 96110391; Ansell et al., Iaurnational PCT Publication No. WO
96/18390; Holland et al., Intornatioaal PCT Publication No. WO 96/10392; all of which are incorporated by reference heroin). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive zo MPS tissues such as the liver and spleen.
The present invention also includes compositions prepared for storage or administration which include ya pbarmac:eutically efr'ective amount of the desired compounds in a pharnraautically ac~~table carrier or diluent. Acceptable carriers or diluents for tlurapeutic use are vv~ell known in the pharmaceutical art, and are described, Zs for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co.
(A.R.
Gennaro edit. 1985) hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents may be provided. These include WO tl0/lT346 PCTNS99I~1t~65 SE
sodium benzoate, sorbic acid aced es~ of p-hydroxybenaoic acid. In addition, antioxidants and suspending agents may be used.
A pbarnnaceuticatly effective dose is that dose required to prav~t, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the s symptoms) of a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the mute of administration, the type of mannmal being treated, the physical characteristics of the spxific mammal under consideration, concurnat medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount betvNxn 0.1 mg/kg and 100 mg/kg body weight/day f o of active ingredients is administered dependent upon potency of the negatively charged polymer.
The nucleic acid molecules of the present invention may also be administered to a patient in combination with other therapeutic compounds to increax the overall therapeutic e$ect. The use of multiple compounds to treat an indication may increase f s the beneficial effects while reducing the presence of side e~'acts.
Alternatively, certain of the nucleic acid molecules of the instant invention (e.g., formula I~ can be expressed within cells from eukaryotic promoters (e.g., Izant and Weinttaub,1985 ScienGc 229, 345; McGacry and Lindquist,1986 Pros. Natl. Acad Set USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad Sci. USA, 88, 10591-5;
Kashani-2o Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992 J.
Yirol, 66, 1432-41; Weerasinghe et aL, 1991 J. Yirol, 65, 5531-4; Ojwang et al., 1992 Proc.
Natl.
Acad Sci. USA 89, 10802-6; Chen et a1, 1992 Nucleic Acids Res., 20, 4581-9;
Sarver et al., 1990 Science 247, 1222-1225; Thompson et al., 1995 Nucleic Acids Res.
23, 2259; Good et al., 1997, Gene Therapy, 4, 45; all of these references are hereby as incorpamted in their totality by rtfe~nce hereia). Those skilled in the art realize that any nucleic acid can be expressed in eulauyotic cells from the appropriate DNA/RNA

WO OOII'134i6 PCTJUS991Z186S

vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et aL, PCT WO 93!23569, and Sullivan et al., PCT WO 94/02595; Ohlcawa et al., 1992 Nteeleie Acids Symp. Ser., 27, 1 S-6;
Taira et-al., 1991, Nucleic Acids Res., 19, S 125-30; Venture et al., 1993 Nucleic Acids Res., 21, s 3249-55; Chowrira et al., 1994 J. Biol. Chem. 269, 25856; all of these references are hereby incorporated in their totality by reference herein).
In another aspect of the invention, RNA molecules of the present inventioa an preferably expressed from transcription units (see for example Couture et al., 1996, TIG., 12, 510) inserted into DNA or RNA v~tors. The recombinant vectors are v o preferably DNA plasmids or viral vectors. ltibozyme exptrssing viral vectors could be constructed based on, but not limited to, adeno-associated virus, rettovinis, adenovirus, or alphavirus. Preferably, the recombinant vectors capable of expressing the nucleic acid molecules are delivered as descrilxd above, and persist in target cells.
Alternatively, viral vectars may be used that provide for transient expression of nucleic ~ s acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecule binds to the target mltNA. Delivery of nucleic acid molecule expressing vectors could be systemic, such as by infiravenous or intra-muscular administration, by administration to target culls ex-planted from the patient followed by, r~introductian into the. gatieut, or by any other means that would allow for . zo introduction into the desired target cell (for a review see Couture et al., I996, TIG., 12, 510).
In one aspect the invention features, an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed. The nucleic acid sequence encoding the nucleic acid molecule is of the instant invention is operable linked in a manner that allows expression of that nucleic acid molecule.

WO OOllT346 PCTNS99/218b5 In another aspect the invention features, an expression vector comprising: a transcription-initiation region (e:g., eukaryotic pal I, II or III initiation region); b) a transcription-ten~nination region (e.g., eukaryotic pol I, II or III
termination region); c) s' gene encoding at least one of the nucleic-acid catalysts of the instant invention; and s wherein said gcne is operably linked to said initiation region and said termination region, in a manor which allows expression and/or delivery of said nucleic acid molecule. The vector may optionally include an open reading frame (ORS for a protein operably linked on the 5' side or the 3'-side of the gene cacoding the nucleic-acid catalyst of the invention; and/or sa intmn (inte<veoing sequences).
~ o Transcription of the nucleic acid n~locule sequences are driven from a promoter for eukaryotic RNA polymerise I (pot I), RNA polymerise II (pol II), or RNA
polymerise III (pol ID). Transcripts from pol II or pol III promoters will be expressed at high levels in all calls; the levels of a given pot II promoter in a given cell type will depend on the nature of the gene regulatory sequences {en>uu~s, silencers, eta) ~ s present nearby. Prokaryotic RNA polymerise promoters are also used, providing that the prokaryotic RNA polymerise enzyme is expressed in the appropriate cells (F.lroy-Stein and Moss, 1990 Pros. Nail. Acid Sel. U S A, 87, 6743-7; Gao and Huang Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993 Methods Fnzymol, 217, 47-66;
Zhou ..yet, al.;. .-1~Q90 -Mol. Cells Biol., 10,. 4529-37)~ Several investigators have -zo demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antfsense Res. lkv., 2, 3-15; O~wang et al., 1992 Proc. Natl. Acid Sci. U S A, 89, 10802-6;
Chen et al., 1992 Nucleic Acids Res., 20, 4581-9; Yu et al., 1993 Proc. Nato Acid.
Sci. U S A, 90, 6340-4; L'Huillier et al., 1992 EMBO J. L 1, 4411-8;
Lisziewicz et al., '. 2s 1993 Proc. Natl. Acid Scl. U. S. A., 90, 8000-4; Thompson et al., 1995 Nucleic Acids Res. 23, 2259; Sullenger & Cech, 1993, Science, 262, 1566). More specifically, transcription units such as the ones derived from genes encoding U6 small anclear we oon~ pcrnJS~t86s b1 (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as riboxyntes in cells (Thonipson er al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acid Res.;
22, 2830; Noonberg et al., US Patent No. 5,624,803; Good et al., 1997, Gene Ther. 4, s 45; Beigelman et al.. Im6ecnationat PCT PubG~tion No. WO 9b/18736; all of these publications are incorporated by referetxre herein. The above ri6ozyme-transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including lxrt not restricted ta, plasmid DNA vxtors, viral DNA vectors (such as adenovirus or adeno-associated vin~a vectors), or viral RNA vectors (s~h as retroviral l o or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
In yet another aspect, the invention features an expression vector comprising a nucleic acid sequence axod>ng at least one of the nucleic acid molecules of the invention, in a meaner which al~ws expression of that nucleic said molecule.
The expression vector comprise in one embodiment; a) a transcription-initiation region; b) l 5 a transcription termination region; c) a gene encoding at least one said nucleic acid molecule; and wherein said gene is openly linked to said initiation region and said termination region, in a manner which allows expression andlor delivery of said nucleic acid molecule. In another preferned embodiment the expression vector comprises: a) a .. _ .. . ... . ~. . ,G~ansaription initiation,region;:b) a transctitption-tenmioation ~gion; c) an open re$ding . .. .
Eo frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is openzbly linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading flame and said termination region, in a manner which allows expression and/or delivery of said nucleic said molecule.
2s In yet another embodbnent, the expression vector comprises: a) a transcription-initiation region; b) a tranxripaoa-termination region; c) an intron; d) a gene encoding at least one said nucleic sad molecule; and wherein said gene is opeiably linked to said wo oon » Pcrrus9sn»s initiation region, said intcnn and said termination region, in a manner which allows expression andlor delivery of said nucleic said molecule.
In a f<uther embodiment, the expression voctor comprises: a) a ttans~iption-initiation region; b) a transcription-teranination region; c) an intron; d) as open reading s frmne; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intmn, said open reading frame and said termination region, in a manner which allows expression andlor delivery of said nucleic acid molecule.
l o Oc~timizinQ Nucleic Acid Molecule Activitv Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases may increase their potency (see e.g., F.ckstein et al., Intaaauional Publication No. WO
92/07065;
Perrault et al., 1990 Nature 344, 565; Pielcen et a!.,1991 Science 253, 314;
Unman and l s Cedergren, 1992 Trends in Biochem. Scf. 1?, 334; Unman et al., International Publication No. WO 93115187; am! ltossi et al., International Publication No.
WO 91/03162; Sproat, US Patent No. 5,334,711; and Hucgin et al., supra: all of these .. ... , . ,~ ~o!~,G#~uca1 m~iH~ons that oaa be madQ to.the base, phosphate andEor . .
sager moieties of the nucleic acid molecules herein). Modifications which enhance Zo their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligoa~leotide synthesis times and reduce chemical requirements are desired.
(All these publications are hereby incorporated by refrrence herein).
'there are several examples in the art describing sugar, base and ~
modifications that can be introduced into nucleic acid molecules with significant 25 CahB~Cilt in their miclease stability and e»cacy. For example, oligonucleotides are modified to enhance stability aadlor enhance biological activity by modification with we oorm3~s rcrrUS~ms6s nuclease resistant groups, for example, 2'-amino, 2'-C allyl, 2'-flouro, 2'~t?-methyl, 2'-H, nucleotide base modifiications (for a review see Unman and Cedergren, 1992 TIES
17, 34; Unman et al., 1994 Nucleic Acids S,ymp. Ser. 31, 1b3; Burgin et al., Biochemistry 35, 14090). Sugar modifications of nucleic acid molecules have been s extensively described in the art (see Eckstein et al., Internatiorurl Publication PCT No.
WO 92/07065; Perrault et a~ Nature 1990, 344, Sb5-5b8; Pieken et al. Science 1991, 253, 314 317; Unman and Cedergren, Treads in Biochem. Sri. 1992, 17, 334-339;
Usman et al. Internatiorzad Publication PCT No. WO 93/1518?; Sproat, US Patent No. 5,334,711 and Beigelman et al., 1995 J. Biol. Clam. 270, 25702; Beigelman et aia, i o International PCT publication No. WO 97126270; Beigelman et al., US Patent No.
5,716,824; Unman et al., US patent No. 5,627,053; Woolf et al., International PCT
Publication No. WO 98/13526; Thompson et al., USSN 60/082,404 which was filed on April 20, 1998; Karpeisky et a~, 1998 Tetrahedron Lett. 39, 1131; all of which are hereby incorporated in their totality by referee l~eram). Such publications, which r s describe general methods and strategies to determine the location of incorporation of sugar, base and/or phosphate modifications and the like into ribozymes without inhibiting catalysis, are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the TiPH
nucleic acid molecules of the instant invention.
to While chemical modification of oligonuclootide iaternueleotick linkages with phasphorothioate, phosphorathioate, and/or 5'-methylphosphonate linkages improves stability, too many of these modifications may cause increased toxicity.
Therefore, when designing HPH molocuks, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity Zs resulting in increased e»cacy and higher specificity of these HPH
molecules.
Nucleic acid molecules having chemical modifications which maintain or enhance activity are disclosed herein. Such nucleic acid is also generally more resistant WO OOJ17346 t'CTNS99I21 ~5 to n~leases than unmodified nureleic acid. Thus, in a cell a~/or in vivo the activity may not be significantly lowered. Therapeutic HPH molecules delivered exogenously must optimally be stable within cells until translation of the target RNA has beeur inhibited long enough to reduce the levels of the undesirable protein. This period of s time varies between hours to days dep~ding upon the disease state. Clearly, nucleic acid molecules raust be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (W'mcott et a~, 1995 Nreettic dcfds Res 23, 2677; Caruthers et al., 1992, Methods in Enrymology 211,3-19) lncarporated by reference herein) have ethe i o ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as gibed above.
Use of these HPH molecules will lead to better treatment of disease progression by affording the possibility of combination therapies (e.g., multiple HFH
molecules targeted to different genes, HPH molecules coupled with known small-molecule i 5 inhibitors, or intermittent treatment with combinations of HPH molecules (including different HPH motifs) and/or other chemical or biological molaarles)). The treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules.
T~et alidation xo One of the most challenging tasks in drug discovery is the choice of a therapeutic target. Historically, traditional biochemical and other studies have offered limited information in this regard. However, recent advances in genomics offer the ' potential to revolutionia both the speed and certainty of therapeutic target identifccation. Progress in charactaiang the genes in the human garome has been very 26 rapid, and it is now estimated that the entire complement of genes in the human genome may be sequaxod before the end of this century. However, this mass of information is coming to the scientific world without a road map. Converting pure gene sequence WO 00/17316 I'CTNS991It865 information into a functional understanding of their role in hcunan disease is pmving to be a much more difficult pmblem. Even after a group of genes is associated with a particular disease, the proctss of validating which genes are appropriate for use as-therapeutic targets is often slow and costly. Mosi companies with genomics activities s now have access to myriad of partial or full sequences, but do not possess adequate technologies to determine which of those sequences is an appropriate therapeutic target.
As a result, only a few genes have been unequivocally identifies as the causative agent for a specific disease.
The nucleic acid molocales of the present invention can inhibit gene ion l o in a highly specific manger by binding to and causing the cleavage of the mRNA
corresponding to the gene of interest, and thereby prevent production of the gene product (Christoffersen, Nature Biotech, 1997, 2, 483-484). Appropriate delivery vehicles can be combined with these nucleic acid molecules (including polymexs, cationic lipids, liposomes and the like) and delivered to appropriate cell culture or in l s vivo animal disease models as described above. By monitoring inhibition of gene expression and correlating with phenotypic results, the relative importance of the particular gene sequence to disease pathology can be established. The process may be both fast and highly selxtive, and allow for the process to be used at any point in the development of the organism. The novel chemical composition of these nucleic acid Zo molecules may allow fog added stability and therefore increased efficacy.
The following are non-limiting examples danonstiatiag the utility of the nucleic acid molecules of the instant invention Those in the art will recogni~c that certain experimental conditions such as temperatures,' reaction times, media conditions, 25 transfection reagegts, cell types and RNA assays are not meant to be limiting and can be readily modified without significantly altering the protocols.

wo oon~~6 pcrnrs~n~ass ss it 'n a The sequences of target RNAs were scxeened for accessible sites using a computer-folding algorithm. Regions of the mRNA that did not form secondary s folding structures were idmtifed. A more elaborate protocol for identifying appropriate targets may be found in Stinchcomb et al., US Pat. No. 5,646,042 which is incorporated by reference in its entirety herein.
k 2: Dowo-Re 'on of~~,~~mssion HPH oligonuclcotides targeting exon 11 of the human c-ref gene, with 4-6 i o complementary nucleotides at the 5' and the 3' end were synthesized using standard protocols (Wincott et al., sxp~a). These 5' and 3' sequencxs were rat complementary to the o-raf target. Of the 23 nucleotides complementary to the target seq~ce, 11 nucleotides in the DNA core and RNA arms were exchanged to ga control molecule which lacks the capability to down-regulate c-ref mRNA in a sequ~o-i s specific manner. The sequences for nucleic acid mokcules used are displayed in table III.
Tissue Culda~e acrd Nucleec Acid delivery Protocol: Prostate cancer cells (PC-3) were gcvwn in a gmwth media consisting of Kaighn's F-12K media, 10~/o FBS, 1%
glutamine, 20 mM HEPF.S, and 1% pedstrep to svb-confluent densitks. A 4X
Zo concentration (10 pg/mL) of GSV (Gkn Research) was preparod from a 2 mg/mL
stock solution as well as a IOpNI solution of the nucleic acid molxule of the present invention and its aatisense control. Complexes of antisensc and GSV were formed in a 96-well plate by channel pipetting in antisense and GSV to form complex solutions which are twice the final concentrations.
Zs hrfiibition of e-ref mRNA lJsir~g Nucleic acid Molecules of Varying Leagtlrs: Using the cell culture and oligonucleotide delivery method described above, PC-3 cells ware treated for 1, 3 or 5 days with lipid-complex~d hairpin oligonucleoddes. The wo oon~~6 Pcrrt~sa~nia6s oligonucleotides used were either 3 I (Seq. LD. No. 12022), 33 (Seq. LD. No.
12021 ), or 35 nucleotides (Seq. 1D. No. 12020) in length. Mismatch controls were used to check for non-specific effects and are given above as Seq. LD. Nos. 12023, 12024, and-12025 for 35, 33, and 3lmers, respectively. An untreated control was also tested to s determine basal levels of c-raf. PC-3 cells wore then harvested with 150 p.L
of RLT
lysis buffer (Qiagen). RNA was purified using Qiagen's instructions and RNA
was quantified using TaqMan'''~ (Perkin Elemer) reagents and the 7700 Prism (P~kin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA over (3-actin mRNA
was determined by real-time PCR after reverse transcription. Results are shown in Fig.
t o 3-5.
The results show that all three molaxeles demonstrate high levels of reduction of c-raf RNA compared to the mismatch controls regardless of oGgonuclootide length.
Inhibition levels ranged from 80-93% in PC-3 cells. After each designated time period, PC-3 cells were harvested with 150 p1, of RLT lysis buffer (Qiagen). RNA was ~ s purified using Qiagen's instructions and RNA was quantified using TaqManT"~ reagents and the 7700 Prism (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA over (i-actin mRNA was deterrained by real-time PCR after reverse transcription.
2o Exg~nle 3: Comt~rlson of c-raf inhibition betvvExo~g"~jlp~rbridizer Molecule and a Linear Antisense Molecule.
To test whether the nucleic acid molecules of the present invention could inhibit c-raf mRNA as well as linear antisense molecules, hairpin and linear antisense molecules were synthesized (Wincott et al., supra). Using the cell culture and 2s oligonucleotide delivery method described in example 2, PC-3 cells were treated for 1,3 or 5 days with lipid-compleaced hairpin oligonucleotides or a lipid complexed linear antisense molecule. The hairpin molecule (Seq. LD. No. 5) was 3! nucleotides in length and the results of c-raf inhibition were compared to a mismatch control (Soq.

wo oon~~ Pcrrtrs9sms6s s~
LD. No. 6). The potency of the antisense molecule was also compared to its mismatch confiol. After each designated time period, PC-3 calls were harvested with 150 pI, of RLT lysis butFer (Qiagen). RNA was purified using Qiagen's instructions and RNA-was quantified using TaqMan"~ reagents and the 7700 Prism (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA over (3-actin mRNA was determined by real-time PCR after revctsc transcription. The data is given in figures 7 and 8. The HPH molecules significantly reduce the c-raf RNA level while the mismatch molecules did not cause any significant reduction (figure 6, 7). Similarly, the linear antisense molecule reduced r-raf RNA levels significantly. These experiments demonstrate that i o the magnitude of c-raf inhibition caused by a hairpin oligonucleotide is comparable with a linear molecule lacldog the 5' and 3' hairpin complem~ ends nle 4: Mutation Analvsia of the Hairnig~vbridizer Molecule The nucleic acid molecules of the present invention were designed to bind to c i s raf message (table II) with 0, 1, 2, or 4 mismatches within the internal DNA sequence.
The molecules were designed such that the 5' sequence is compleme~ry to both the 3' sequence as well as the target molecule. These molecules were delivered to PC-3 cells using the cell culture and aligonucleotide delivery protocol, described in example 2.
The lipid/nucleic acid molecule complexes were added to the cells and allowed to ao associate for 24 hours. PC-3 cells were then harvested with 150 PI. of RLT
lysis buffer (Qiagen). RNA was puri$ed using Qiagen's idons and RNA was quantified using laqMan?'~ reagents and the 7700 Prism (Perlcin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA over ~-actin mRNA was determined by real-time PCR after reverse transcription. The results are shown in figure 9. Just a single as mutation within the HPH nucleic acid molecute is sufficient to destroy the inhibitory effects of the HPH molecule. This shows that the HPH molecules of the present invention are highly sequ~oe-specific reagents.

wo oorm pcrnrs99ntsbs Examg~e 5: Inhibition of IMDPH II RNA ~~~i p. Wij~~y~ridizinQ
Molecuies of Varying Lengths.
Prostate cancer cells (PC3) were grown as described above. Nucleic acids were complexed and applied to cells as described, with the exception that a cationic lipid was s used. The final oligonucleotide concentration was 100 nM. RNA levels were measured by TaqMan''"' analysis as described above.
Using the cell culture and oligonucleotide delivery method described above, cells were treated for 24 hours with lipid-complexed oligonucleotides. The oligonucleotides targeted to IMPDH II had; in one case, a 23mer target-hybridizing r o region plus a 6 base hairpin hybridizing region at the 3' end that annealed to the 5' end (Seq. ID No.l l, and 2 base mismatch control Seq. ID No. 12). Alternatively, the oligonucleotide had a l9mer target-hybridizing region plus a 4 base hairpin-hybridizing region at the 3' end that annealed to the 5' end (Seq. ID No. 13, and 2 base mismatch control Seq. ID No. 14). As shown in Fig. 12, the hairpin hybridizer molecule a s demonstrated 45-70~/o inhibition of the target RNA level relative to untreated cells. In both cases a 2 base mismatch was sufficient to prevent target down-regulation, demonstrating the high specificity of these reagents.
Example 6: Alternative Hairuin A~i», Domains C~~fer Cornparablo Efftcac~
2o Using the cell culture and oligonucleotide delivery method described above, cells wire treated for 24 hours with lipid-complexed oligonucleoti~s. The oligonucleotides targeted c-Raf exon 11, and consisted of DNA core regions (an example of RNase H-activating Region) at or near the 5' end of the oligonuclootide, and a 3' hairpin hybridizing regions that could anneal to different regions of the target-2s complementary region, including the DNA core and/or the RNA arms. As shown in Fig. 11, a 2lmtr target-hybridizing region with a 6 base hairpin that anneals to the 5' end of the oligo overlapping part of the DNA core (Seq. ID No. I 5) shows greater than 80% inhibition of target RNA expression. Scrambling the 3' end to prevent formation of an intramolecular hairpin (Seq. ID No. 16) neither enhances nor interferes with the 3o cell efficacy in this assay, indicating that the oligonucleotide may lx able to basepair to the target RNA equivalently with or without the hairpin structure. An l8mer target-wo oon~~s . Pcrnrsr~msss ~o hybridizing (complementary) region with varying 6 nucleotide self-complementary struct<u~es (Seq. ID Nos. 17, 18, 19, 20) shows 35-65% inhibition of target RNA
expreSSion, indicating that a variety of stnxhues can result in e~cacious molecules The difference in magnitude between the 2lmer and l8mer structures probably reflects s the lower target binding ai~nity of the l8mer. The differences in magnitude of inhibition between Soq. ID Nos. 17, 18, 19, 20 may reflect subtle sequence dependence variations in the optimal structure.
Using the cell culture and oligonucleotide delivery method described above, cells wets treated for 24 hours with lipid-complexed oligon~leotides. The l o oligonucleotides targeted IMPDH II, and consisted of DNA core regions in the center of the oligonucleotide, and a 3' hairpin hybridizing regions that could anneal to the DNA core. As dawn in Fig. 13, a linear antisenae oligoawcleotide with a 23mer target-hybridizing region (Seq. ID No. 22) gave 70% inhibition, while random sequence and scrambled sequence negative controls (Seq. ID No. 21, 23) gave virtually no inhibition.
~ s Hairpin oligos (Sal. ID No. 24, 25) with either a 4-or a 6-base hairpin that anneals to the DNA core showed 60-70% inhibition of target RNA expression. In another example, a linear oligo targeting a different site in the target RNA (Seq. ID
No. 2~
gave 80% inhibition, and hairpin oligos {Seq. ID Nos. 27, 28) gave 70-80%
inhibition of target RNA expression.
2o Those skilled in the art will recognize that the instant invention is not limited to the HPH molecules used in the foregoing examples. The instant invention broadly features HPH~ oligonucleotides of varying structures, including those hybridizing to an internal RNase H-activating regions, hybridizing to both the RNase H-activating region and the Non-RNase H-activating region, and those hybridizing to a RNase H
activating 2s region at either the 5' or 3' end (for example see Figures 1-3 and 10).
The hairpin structure could provide protection against exonucleolytic andlor endonueleolytic degradation, thus increasing stability both in vivo and en vitro. The hairpin creates a duplex region that juxtaposes various chemical end-modifications that may confer altered in vivo pharmacokinetics or tissue distribution. Thus these 3o molecules may have advantages compared to tr~litional linear antisense molecules for use as therapeutics or as tools for in vivo target validation.

Dia~ctnostic uses Nucleic acid molecules of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of s specific RNAs in a cell. The close relationship between antisense activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alttrs the baso-pairing and three-dimensional structure of the target ~A. By using multiple nucleic acid molecules described in this invention, one may map nucleotide changes which are important to RNA structure and function in vitro, as i o well as in cells and tissues. Inhibition of target RNAs with nucleic acid molecules may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disuse. These experiments will lead to better treatment during disease progression by affording the possibility of combinational i s therapies (e.g., multiple nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of nucleic acid molecules and/or other chemical or biological molecules). Other in vitro uses of nucleic acid molecules of this invention are well .. . . ,, , ~ , down in the art, and include detection of the pre~ce of RNAs related to various . , , zo conditions.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains.
All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
25 One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well we oon~~s pcrnJS~ma6s n as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to' those skilled in the art, which are encompassed within the spirit of the invention, are s defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present invention and the following claims.
t o The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the tens "comprising", "consisting essentially of and "consisting of' may be replaced with either of the other two terms. The terms and expressions which have been employed t s are used as terms of description and not of limitation, and there is no intention that in the use of such teens and expressions of excluding any equivalents of the futures shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred Zo embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms of 2s Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or wo oon~~s pcrnrs9nmabs subgroup of members of the Markush group or other group.
Otlur embodiments arc within the following claims.

wo oonr~s rcrius~msss l4 Table I: D.2 pmol RNA Synthesis Cycle Reagents fiquivalentsAmounts ~L) Wait tune (sec) -Phosphoramidites15 31 465 SET 38.7 31 465 Acetic anhydride655 124 5 N-methyl- 1245 i24 5 imidazole TCA 700 . 732 10 Iodine 20.6 244 15 * Wait time does not include contact time during delivery.

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Claims

1. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence expression, wherein said hairpin hybridizer nucleic acid molecule consists of the formula I:

wherein, each P, Y, N, and M represents independently a nucleotide which may be the same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides; N' is a nucleotide complementary to N; o is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k and t are independently zero or an integer greater than or equal to 3; wherein when t or k is independently 3 or greater, at least one said P is not a 2'-H containing nucleotide; each said (P)t and (P)k includes internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M)w is an oligonucleotide including one or more internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate, and phosphorodithioate linkage; wherein at least one or more of each said (P)t, (P)k, and (M)w is an oligonucleotide is of sufficient length to stably interact with the target sequence; r and f are independently an integer greater than or equal to zero; each B and B' independently represent, a cap structure which may independently be present or absent; and ~
represents a chemical linkage.
2. The method of Claim 1, wherein k in said hairpin hybridizes nucleic acid molecule is less then 100.
3. The method of Claim 2, wherein, k in said hairpin hybridizes nucleic acid molecule is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
4. The method of claim 1, wherein, t in said hairpin hybridizes nucleic acid molecule is less than 100.
5. The method of claim 4, wherein, t in said hairpin hybridizes nucleic acid molecule is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
6. The method of claim 1, wherein, k and t in said hairpin hybridizer nucleic acid molecule are of the same length.
7. The method of claim 1, wherein, k and t in said hairpin hybridizer nucleic acid molecule use of different lengths.
8. The method of claim 1, wherein w in said hairpin hybridizes nucleic acid molecule is less than 100.
9. The method of claim 8, wherein w in said hairpin hybridizes nucleic acid molecule is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
10. The method of claim 1, wherein the t, k, and w in said hairpin hybridizer nucleic acid molecule are of same length.
11. The method of nucleic acid molecule of claim 1, wherein t, k, and w in said hairpin hybridizer nucleic acid molecule are of different length.
12. The method of claim 1, wherein the target sequence is selected from the group consisting of RNA, DNA and RNA/DNA mixed polymers.
13. The method of claim 1, wherein r and f in said hairpin hybridizer nucleic acid molecule are independently selected from the group consisting of 1, 2, 3, 4, 5, 10, and 15.
14. The method of claim 1, wherein the chemical linkage in said hairpin hybridizer nucleic acid molecule is selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiopohisphate, methylphosphonate, and phosphorodithioate.
15. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of the formula II:

wherein, each P, N, and M represents independently a nucleotide which may be same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; o is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k, t, k1 and t1 are independently zero or an integer greater than or equal to 3; wherein when t, k, t1, or k1 are independently 3 or greater at least one said P is not a 2'-H
containing nucleotide; each said (P)t, (P)k, (P)t1, and (P)k1 independently include internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M)w is as oligonucleotide sequence including one or more internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate, and phosphorodithioate linkers; wherein at least one or more of each said (P)t, (P)k, (P)t1, (P)k1 and (M)w is an oligonucleotide of sufficient length to stably interact independently with the target sequence; and ~ represents a chemical linkage.
16. The method of claim 15, wherein each k, t, k1, t1 and w in said hairpin hybridizer molecule is less than 100.
17. The method of claim 16, wherein each k, t, k1, t1 and w in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
18. The method of claim 15, wherein k and t in said hairpin hybridizer nucleic acid molecule are of same length.
19. The method of claim 15, wherein k and t in said hairpin hybridizer nucleic acid molecule are of different length.
20. The method of claim 15, wherein t, k, and w in said hairpin hybridizer nucleic acid molecule are of the same length.
21. The method of claim 15, wherein t, k, and w in said hairpin hybridizer nucleic acid molecule are of different length.
22. The method of claim 15, wherein t1, k1, and w in said hairpin hybridizer nucleic acid molecule are of the same length.

23. The method of claim 15, wherein t1, k1, and w in said hairpin hybridizer nucleic acid molecule are of different length.
24. The method of claim 15, wherein the target sequence is selected from the group consisting of an RNA, DNA and RNA/DNA mixed polymer.
25. The method of claim 15, wherein said chemical linkage in said hairpin hybridizer nucleic acid molecule is selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophosphate, methylphosphonate, and phosphorodithioate.
26. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula III:

wherein, each P, N, and M represents independently a nucleotide which may be same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; o is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k and t independently are zero or an integer greater than or equal to 3; wherein when t and k are independently 3 or greater at least one said P is not a 2'-H containing nucleotide; each said (P)t and (P)k includes internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M)w is an oligonucleotide sequence including on or more internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate and phosphorodithioate; D and E are oligonucleotides independently of length greater than or equal to 4; wherein at least one or mote of each said (P)t, (P)k, (M)w, D and E are independently an oligonucleotide of sufficient length to stably interact independently with a target nucleic acid sequence; B and B' independently represent a cap structure which may be present or absent; and _____ represents a chemical linkage.

27. The method of claim 26, wherein each k, t, and w in said hairpin hybridizer nucleic acid molecule is independently less than 100.

28. The method of claim 27, wherein each k, t, and w in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 4, 5, 6, 7, 8, 9,10,11,12,15, and 20.

29. The method of claim 26, wherein k and t in said hairpin hybridizer nucleic acid molecule are of the same length.

30. The method of claim 26, wherein k and t in said hairpin hybridizer nucleic acid molecule are of different length.

31. The method of claim 26, wherein each t, k, and w in said hairpin hybridizer nucleic acid molecule ace of the same length.

32. The method of claim 26, wherein each t, k, and w in said hairpin hybridizer nucleic acid molecule are of the same length.

33. The method of claim 26, wherein the target nucleic acid sequence is selected from the group consisting of RNA, DNA and RNA/DNA mixed polymer.

34. The method of claim 26, wherein each D and E oligonucleotides is independently less than 100 nucleotides in length.

35. The method of claim 26, wherein the length of each D and E
oligonucleotides is independently selected from the group consisting of 6, 7, 8, 9, 10, 11, 12, 15, 20, and 30 nucleotides.

36. The method of claim 26, wherein D and E oligonucleotides in said hairpin hybridizer nucleic acid molecule are of the same length.

37. The method of claim 26, wherein said oligonucleotides, said D and said E
oligonucleotides in said hairpin hybridizer nucleic acid molecule are of different length.

38. The method of claim 26, wherein said chemical linkage is selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, and phosphorodithioate.

39. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula V:
wherein, each P, N, F, V, Z, and M represents independently a nucleotide which may be same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3; w is an integer greater than or equal to 4; d is an integer greater than or equal to 3; h is an integer greater than or equal to 2; c is an integer greater than or equal to 0; k and t are independently, zero or an integer greater than or equal to 3; wherein when t and k are independently 3 or greater, at least one said P is not a 2'-H
containing nucleotide; each (P)t,(P)k, (V)d and (Z)c includes internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M)w is an oligonucleotide including one or more internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate and phosphorodithioate linkage; wherein at least one or more of each said (P)t, (P)k, and (M)w is an oligonucleotide of sufficient length to stably interact independently with a target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

40. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula VI:
wherein, each P, N, F, Z and M represents independently a nucleotide which may be same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3; k and t are independently, zero or an integer greater than or equal to 3; k1 and t1 are independently zero or an integer greater than or equal to 3; w is an integer greater than or equal to 4; h is an integer greater than or equal to 2; c is an integer greater than or equal to 0; wherein when t, k, t1, or k1 is independently 3 or greater at least one said P is not a 2'-H containing nucleotide; each (P)t,(P)k, (P)t1,(P)k1, and (Z)c includes internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M)w is an oligonucleotide including one or more inter-nucleotide linkerages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate and phosphorodithioate linkage; wherein at least one or more of each said (P)t, (P)k, (P)t1,(P)k1, and (M)w is an oligonucleotide of sufficient length to stably interact independently with a target nucleic acid molecule; each B and B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

41. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula VII:

wherein, each P, N, F, V, Z, and M represents independently a nucleotide which may be same or different; .cndot. indicates hydrogen bond formation between two adjacent nucleotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3; w is an integer greater than or equal to 4; d is an integer greater than or equal to 3; h is an integer greater than or equal to 2; c is an integer greater than or equal to 0; k and t are independently, zero or en integer greater than or equal to 3; wherein what t and k are independently 3 or greater, at least one said P is not a 2'-H
containing nucleotide; each (P)t ,(P)k, (V) d and (Z)c includes internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'thiophosphate, and methylphosphonate; (M]w is an oligonucleotide including one or more internucleotide linkages selected from the group consisting of phosphodiester, phosphorothioate, 5'thiophosphate, methylphosphonate and phophorodithioate linkage; wherein at least one or more of each said (P)t, (P)k, and (M)w is an oligonucleotide of sufficient length to stably interact independently with a target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and _____ represents a chemical linkage.

42. The method of any of claims 39-41, wherein each k, t, and w in said hairpin hybridizer nucleic acid molecule is independently less than 100.

43. The method of claims 42, wherein each k, t, and w in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 4~, 5, 6, 7, 8, 9,10,11, 12,15, and 20.

44. The method of any of claims 39 or 41 wherein d in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 3, 4, 5, 6, 7, 8, 9,10, 11,12, 16, and 18.

45. The method of any of claims 39-41, wherein h in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 2, 3, 4, 5, 6,7,8, and 9.

46. The method of any of claims 39-41, wherein c in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11,12, 16, and 18.

47. The method of any of claims 39-41, wherein o in said hairpin hybridizer nucleic acid molecule is selected from the group consisting to 4, 5, 6, 7, 8 and 9.

48. The method of any of claims 39-41, wherein k and t in said hairpin hybridizer nucleic acid molecule are of the same length.

49. The method of any of claims 39-41, wherein k and t in said hairpin hybridizer nucleic acid molecule are of different length.

50. The method of any of claims 39-41, wherein each t, k, and w in said hairpin hybridizer nucleic acid molecule are of different length.

51. The method of any of claims 39-41, wherein each t, k, and w in said hairpin hybridizer nucleic acid molecule are of the same length.

52. The method of any of claims 39-41, wherein the target sequence is selected from the group consisting of RNA, DNA and RNA/DNA mixed polymer.

53. The method of any of claims 39-41, wherein said chemical linkage is selected from the group consisting of phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophosphate, methylphosphonate, and phosphorodithioate.

54. The method of claim 40, wherein each k1 and t1 in said hairpin hybridizer nucleic acid molecule is independently less than 100.

55. The method of claim 40,wherein each k1 and t1 in said hairpin hybridizer nucleic acid molecule is independently selected from the group consisting of 4, 5, 6, 7, 8, 9, 10,11, 12, 15, and 20.

56. The method of claim 40,wherein each t1, k1, and w in said hairpin hybridizer nucleic acid molecule are of different length.

57. The method of claims 40,wherein each t1, k1, and w in said hairpin hybridizer nucleic acid molecule are of the same length.

58. The method of any of claims 39-41, wherein said F portion of the (F.cndot.F')k in the HPH nucleic acid molecule is complementary to a portion of said target sequence.

59. The method of any of claims 39-41, wherein said F' portion of the (F.cndot.F')h in the HPH nucleic acid molecule is complementary to said target sequence.

60. The method of any of claims 39-41, wherein said F and said F' portion of the (F.cndot.F')k in the HPH nucleic acid molecule is independently complementary to said target sequence.

61. The method of any of claims 1, 15, 26, or 39-41, wherein said N portion of the (N.cndot.N') in the HPH nucleic acid molecule is complementary to a portion of said target sequence.

62. The method of any of claims 1, 15, 26, or 39-41, wherein said N' portion of the (N.cndot.N') in the HPH nucleic acid molecule is complementary to said target sequence.

63. The method of any of claims 1, 15, 26, or 39-41, wherein said N and said N' portion of the (N.cndot.N')o in the HPH nucleic acid molecule are independently complementary to said target sequence.

64. The method of any of claims 39-41 wherein, said (Z), in the HPH nucleic acid molecule is complementary to a target sequence.

65. The method of any of claims 1, 15, 26, or 39-41, wherein each said (P)k, (P)t, (N.cndot.N')o, and (M)w, in the HPH nucleic acid molecule comprises independently a nucleotide modification selected from the group consisting of: 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol;
2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl) amino; 2'-(N-phenylalanyl)amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide;
and xylofuranosyl.

66. The method of claim 1, wherein each said (Y)r and (Y)f in the HPH nucleic acid molecule comprises independently a nucleotide modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl)amino; 2'-(N-phenylalanyl)amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide; and xylofuranosyl.

67. The method of claim 15 or 40 wherein each said (P)k1, and (P)t1, in the HPH
nucleic acid molecule comprises independently a nucleotide modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl) amino; 2'-(N-phenylalanyl)amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide;
and xylofuranosyl.

68. The method of claim 26, wherein each said D and E in the HPH nucleic acid molecule comprises independently a nucleotide modification selected from the_ group consisting of: 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl) amino; 2'-(N-phenylalanyl)amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide; and xylofuranosyl.

69. The method of any of claims 39-41, wherein each said (Z) c, and (F.cndot.F')~, in the HPH nucleic acid molecule comprises independently a nucleotide modification selected from the group consisting of: 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl) amino; 2'-(N-phenylalanyl) amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide;
and xylofuranosyl.
70. The method of any of claims 39 or 41 wherein each said (V) d, in the HPH
nucleic acid molecule comprises independently a nucleotide modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methylthiomethyl, L-nucleotides; 2'-C-allyl; 1-5-Anhydrohexytol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2'-(N-alanyl) amino; 2'-(N-phenylalanyl)amino; 2'-deoxy-2'-(N-b-alanyl) amino; 2'-deoxy-2'-(lysyl) amino; 2'-O-amino; 2'-Deoxy-2'-(N-histidyl) amino; 6-methyl uridine; 5-methyl cytidine; 2'-(N-b-carboxamidine-b-alanyl) amino-2'-deoxy-nucleotide;
and xylofuranosyl.
71. The method of any of claims 1, 26, or 39-41, wherein said B' when present, selected from the group consisting of: inverted abasic residue; 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; and methylphosphonate moiety.
72. The method of any of claims 1, 26, or 39-41, wherein said nucleic acid molecule comprises a 3'-3' linked inverted abasic moiety at said 3' end.
73. The method of any of claims 1, 26, or 39-41, wherein said B when present, is selected from the group consisting of: 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate;
hydroxypropyl phosphate; 1-5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5-phosphoramidate; 5'-phosphorothioate; 1,4-butanodiol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties.
74. The method of any of claims 1, 15, 26, or 39-41 wherein said cell is a mammalian cell.
75. The method of any of claims 1, 15, 26, or 39-41 wherein said cell is a plant cell.
76. The method of any of claims 1, 15, 26, or 39-41 wherein said cell is a bacterial cell 77. The method of any of claims 1, 15, 26, or 39-41 wherein said cell is a microbial cell.

78. The method of any of claims 1, 15, 26, or 39-41 wherein said cell is a fungal cell.

79. The mammalian cell of claim 74, wherein said mammalian cell is a human cell.

80. The method of any of claims 1, 15, 26, or 39-41, wherein said HPH nucleic acid molecule is chemically synthesized.

81. The method of any of claims 1, 15, 26, or 39-41, wherein said HPH is a pharmaceutical composition.

82. The method of any of claims 1, 15, 26, or 39-41, wherein said modulation of function is the modulation of the phenotype of the cell.

83. A method of modulating the function of a target sequence in a cell comprising the step of contesting said cell with a hairpin hybridizes (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizes nucleic acid molecule consists of formula IX:

wherein, each F, D, O, K, W and T represents independently an oligonucleotide where nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioste, and methylphosphonate; F and D

independently form a RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D
comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; the K.cndot.T and O.cndot.D base-paired regions within the HPH
nucleic acid molecule may be contiguous or non-contiguous to each other; K, T, O, and W
are of length greater than or equal to 3 nucleotides; F, D, K, T, W and O
together are of sufficient length to stably interact with the target sequence;
each B and B' independently represents a cap structure which may independently be present or absent; and _____ represents a chemical linkage.

84. A method of modulating the function of a target in a cell comprising the step of contacting said cell with a hairpin hybridizes (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizes nucleic acid molecule consists of formula X:

wherein, each F, D, O. K. W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D
comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; the K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other; K, T, O, and W are of length greater than or equal to 3 nucleotides; F, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and _____ represents a chemical linkage.

85. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XI:

wherein, each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently or in combination form RNaseH-activating region; .cndot.
indicates hydrogen bond formation between two adjacent nucleotides within the HPH
nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
K
and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; the K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other, K, T, O and W are of length greater than or equal to 3 nucleotides; F, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and _____ represents a chemical linkage.

87 A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XII:

wherein, each D, O and W represents independently an oligonucleotide whose nucleotide sequence may be same at different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequetart of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; O and W are of length greater than or equal to 3 nucleotides;
D, W and O together are of sufficient length to stably interact with the target sequerare; each B and B' independently represents a cap structure which may independently be present or absent; and _____ represents a chemical linkage.

86. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XIII:

wherein, each D, O and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; O and W are of leagth greater than or equal to 3 nucleotides;
D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

87. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizes (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XIV:

wherein, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length greets than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

88. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XV:

wherein, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complemartary to the nucleotide sequence of T; D act O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length greater than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

89. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XVI:

wherein, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic said molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other, A, K, T, O and W are of length greater than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B sad B' independently represents a cap structure which may independently be present or absent; and ______ represents a chemical linkage.

90. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XVII:

wherein, each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; K.cndot.T and O.cndot.D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length greater than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

91. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizes nucleic acid molecule consists of formula XVIII:

wherein, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D
independently forms as RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; A, O and W are of length grater than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

92. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XIX:

wherein, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; A, O and W are of length greater than or equal to 3 nucleotides; A; D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

93. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XX:

wherein, each F, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently form an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; O and W are of length greater than or equal to 3 nucleotides; F,D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

94. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XXI:

wherein, each F, D, O, and W repress independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently or in combination form an RNaseH-activating domain; .cndot.
indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form grater than or equal to two base pairs with each other that are contiguous or non-contiguous;
O and W are of length greater than or equal to 3 nucleotides; F, D, W and O
together are of sufficient length to stably interact with the target sequence;each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

95. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XXII:

wherein, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; A, O and W are of length greater than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

96. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XXII:

wherein, each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D
independently forms an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; A, O and W are of length greater than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

97. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XXVI:

wherein, each F, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently or in combination form an RNaseH-activating domain; .cndot.
indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous;
O and W are of length greater than or equal to 3 nucleotides; F, D, W and O
together are of sufficient length to stably interact with the target sequence;
each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

98. A method of modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the modulation of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule consists of formula XXV:

wherein, each F, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and include individually or in combination, internucleotide linkage selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D
independently form an RNaseH-activating domain of length greater than or equal to 4 nucleotides; .cndot. indicates hydrogen bond formation between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D
and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous; O and W are of length greater than or equal to 3 nucleotides; F, D; W and O together are of sufficient length to stably interact with the target sequence; each B and B' independently represents a cap structure which may independently be present or absent; and ~ represents a chemical linkage.

100. A method of inhibiting the function of a target sequence in a cell, wherein said target sequence is encoded by c-raf gene, comprising the step of contacting said call with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the inhibition of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule comprises any of sequence selected from the group consisting of Seq ID Nos 1,15, 17, 18, 19 and 20.

101. A method of inhibiting the function of a target sequence in a cell, wherein said target sequence is encoded by IMPDH II gene, comprising the step of contacting said cell with a hairpin hybridizer (HPH) nucleic acid molecule under conditions suitable for the inhibition of said target sequence function, wherein said hairpin hybridizer nucleic acid molecule comprises any of sequence selected from the group consisting of Seq ID Nos 11, 13, 24, 25, 27 and 28.