CA1319119C - Nucleic acid chelate conjugate as therapeutic and diagnostic agents - Google Patents

Abstract

NUCLEIC ACID CHELATE CONJUGATE
AS THERAPEUTIC AND DIAGNOSTIC AGENTS

ABSTRACT OF THE DISCLOSURE
Novel conjugates are provided comprising, in particular, stable nucleotide sequences joined to a chelating agent. The conjugates find use in inhibiting expression of mRNA, in killing pathogens, and in providing selection for particular classes of cells or killing particular classes of cells. Polyamine, polycarboxylic acid, chelating agents are employed in the conjugates as exemplary.

Description

131~1 19 llUCLEIC ACID CXELAl`E CONJUGATE~
J~S THER~PEUTIC AND DIAGNOSTIC A(;ENTS

The field concerns the u~e of modi~ied oligo-nucleotides as therapeutic agents, inhibiting matura-tion or expression of transcrLption products.in vivoand in vitro.

The biological reYolution ha~ introduced a 15 ~arlety of new technique~ resulting ln the abilLty to determine Yarious cellular and subcellular proces3es.
. As the understanding has increased a3 to how the cell j maintains its viability and proli~erates, new opportu-. nities have opened ~or utilizing novel therapeutic 20. approaches. One technique which has been u~ed in a variety of ways in the laboratory and i~ being expanded ¦ from the laboratory into real life s1tuations, i9 the ¦ use of anti-~ense sequences to modulate the fate o~ a transcription product in a host cell. ~.
For the mo~t part, the ap~roaches in employing anti-sense ~eq,uences have been t~o~old. In one appr~oach, cells in culture are modified by inJection o~
a large excess of a DNA sequence which i5 comple~entary to the sequence o~ a mRNh present in the cell. A ~ub-~tantial reduction in the expression product is obser~ed. In another approach, one can lntroduce a tranqcription caYsette comprising a promoter ~unctional in the host and a DNA ~equence which result~ in the production of a mRNA which is complementary to an endo-:. 35 genous mRNA. Again, one obserYe~ a reduction in the : expres3ion product to which the transcription cons~ruc~
',j ia d~rected.

;, ~ .

If, however, anti-sense ~equences are to become a useful therapeutic agent, there are many pro-blems and difficulties to be overcome. As a therapeu-tic agent, a method must be found which allows for the transfer of the anti-sense sequence across the cellular membrane. The anti-sense sequence must be designed so as to be relatively stable to degradation, particularly by nucleases. In addition, there remain concerns about the specificity of the sequence, particularly where one does not wish to kill the host cell. Also o~ concern is how to reach the necessary concentration in the cell to provide the desired level of inhibition of tran-scription product maturation or expression. Finally, any technique which is devised for ~ulfilling the above objectives, must take into consideration such problems as toxicity, immunogenicity, solubility, and the like.
There is, therefore, substantial interest in being able to develop anti-sense sequences which will be effective as therapeutic agents in the modulation of the forma-tion of mRNA and its expression.

Relevant Literature Anti-sense nucleic acid sequences have been reported to selectively block translation of a number 25 of mRNAs (Izant and Weintraub, Cell (1984) 36:1007-1015; Izant and Weintraub, Science (1985) 229:345-352;
Melton, ~roc.~Natl. Acad Sci. USA (1985) 82:144-148; ~-; Mizuno et al. 9 Proc. Natl. Acad. S_i. USA (1984) 81:1966-1970). Short oligonucleotides have been 3 reported to provide for high selectivity. (Wallace et al., Nucl. Acids Res. (1979) 6:3543-3557; Wallace et al., Nucl. Acids Res. (1981) 9:879-894; Smith, et al., _ Proc. Natl._Acad. Sci. USA (1986) 83:2787-2791;
Szostak, et al., Methods Enzymol. (1979) 68:419~429; Wu et al., Prog. Nucl. Acid Res. and Mol. Biol. (1978) ; 21:102). The short oligonucleotides are able to rapidly and peci~ically bind to speci~ic target 3 ~3191 19 sequences. (Itakura and Rlggs, Science (1980) 209:
1401; Szostak et al., Methods Enzymol. (1979) 68:419-429; Noyes et al., J. Biol. Chem. (1979) 254:7472-7475;
Noyes et al., Proc. Natl Acad. Sci. USA (1979) 76:1770-5 1774; Agarwal et al., J. Biol. Chem. (1981) 256:1023-1028; Tullis et al., Biochem. Biophys. Res. Comm.
(1980) 93:941). See also the use of oligonucleotide probeq to detect point mutations. (Orkin et al., J.
Clin. Invest. (1983) 71:775; Conner et al., Proc Natl.
10 Acad. Sci. USA (1983) 80:278; Piratsu et al., New Eng.
J. Med. (l983) 309:284-287; Wallace et al., Nucl.
Acids. Res. (1981) 9:879-894). The rapidity with which -synthetic DNAs hybridize is related to the complexity of the~probe. (Wetmur and Davidson, J. Mol. Biol.
(1967) 31:349; Wallace et al., Nucl. Acids Res. (1979) 6:3543-3557; Tullis et al., Bioche_. Bio~y~
Comm. (1980) 93:941; Meinkoth and Wahl, Anal. Biochem.
(1984) 138:267-284~.
Both normal and phosphorous modified oligonu-cleoides have been reported to selectively block the expression of specific RNAs. Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA (1978) 75:280-284; Tullis et -al., J. Cellular Biochem. Suppl. (1984) 8A:58 (Abstract); Kawasaki, Nucl. Acids Res. (1985) 13:4991;
Haeptule et al., Nucl. Acids R_s. (1986) 14:1427-1448;
~alder et al., Science August 1, 1986; Stephenson and Zamecni~, Proc. Natl. Acad. Sci. USA (1978) 75:285-288 Cornelissen et al., Nucl. Acids Res._ ~1986) 14:5605-5614; Minshall and Hunt, Nucl. Acids Res. (1986) 3 14:6433-6451).
Protection from nuclease degradation can be achieved by employing phosphotriesters and methylphos-phonates. (Barrett et al., Biochemistry (1974) 13:
4897-4906; Miller et al., Biochemiqtry (1977) 16:1988-- 35 1997; Miller et al., Biochemistry (1981) 20:1873-1880;
; Jayaraman et al., Proc. Natl. Acad~ Sci. USA (1981) 77:1537-1541; Blake et al., Biochemistry (1985) 24:6132-6138; 81ake et al., Biochemiqtr~ (1985) 24:
6139-6145; Smith et al., Proc. Natl. Acad._Sci. USA
(1986) 83:2787-2791; Agrls et al., Biochemistry (1986) 25:6268-6275; Miller st al., Nuci. Acids. Res. (1983) 5 11 :6225-6242) .
A number of group~ have tried to enhance the binding affinity by modifying the nucleotide ~equence.
(Summerton, J. Theor. Biol. (1978) 78:77-99; Knorre et al., Adv. Enz. Reg. (1984) pp . 277 -300; ~la~ov et al., 0 Adv. Enz. Reg. (1986) pp. 301-320).

Oligonucleotide con~ugates are provided, where a specific sequence of at least eight nucleotide3 i~
15 covalently linked to an ion chelating group and option-ally to other groups to enhance tran~port across the cell membrane. The resulting composition~ are found to effectively block the function o~ a sequence comple-mentary to the oligonucleotide. The compo~ition3 find use a~ agentq in vivo and in vitro for modulating intracellular tranqcription product maturation or expression.

Method3 and compositions are provided ~or modulating transcriptional maturation or expression by employing oligonucleotide conjugate~. The oligonucleo-; tide con~ugate~ have at least two components: an oli-gonucleotide qequence Or at least eight nucleotide~;
3 and a chelating agent. In addition~ other groups may be preqent which include a linker ~oining the chelating agent to the oligonucleotide sequence, a hydrophobic group ror enhancing the transport acro~ the membrane, or other moietieq to enhance binding af~inity, reduce ~ 35 toxicity, enhance solubilityg or other characteristias :. Or intere~tO

1 ~1 ql 1 9 The sub~ect CompositiOns will generally have a hybridizing sequence, namely a polynucleotide unit from about 8 to 30, more usually from about 8 to 20, prefer-ably from about 12 to 18 members. The molecular weight will normally be under about 10 kD, usually under about 6 kD, although high molecular weight moleCUles may be used in special circumstances. The chelating agent may be any one of a large number of chelating agents which are able to chelate metal ions capable of acting as scis~ile and/or free radical initiating agents, by themselVes or in conjunction with other compounds which may be present in the host cell or introduced in the host cell. The chelating agent will be able to chelate one of a variety of metals, such as iron, cobalt, nickel, molybdenum, vanadium, or other metal Lon whiCh may be encountered in the cytoplasm of the cell and may serve to initiate the formation of free radicals, resulting in the scission or other modification of the transcription product, preventing its normal function in the host cell.
For the most part, the compo~itions of the subject invention will have the following formula:

{ ¦ N ~ N a ! N jb }
. Wherein:
K represents the chelating agent capable of chelating a metal ion, which ion is capable of catalyz-ing a chemical reaction in the phy9iological medium of the cytoplasm of a cell, which re9ult9 in a chemical transformation of mRNA inhibiting expression, particu-larly degradative modification;
L is a bond or linking unit derived from a ; 35 polyvalent functional group having at least one atom, which functional sroup may be of from about 1 to 20 atoms other than hydrogen, compri~ing carbon, nitrogen, 6 1;~191 19 oxygen, sulfur, and pho~phorous, where the linking group may be aliphatic, aromatic, alicyclic, hetero-cyclic, or combinations thereof; substituted or unsub-stituted; generally having-from 0 to 10 heteroatoms, usually from 0 to 6 heteroatoms, where the cyclic com-pounds will usually have from 1 to 2 ring~, usually 1 ring, and the aliphatic groups may be branched, straight chained, heterosubstituted or unsubstituted;
desirably the linking unit will have a chain o~ 2 to 10 20, usually 4 to 16 atoms normally free of linkages capable of enzymatic degradation; L may be ~oined through Y to the terminal phosphorous or may be joined at any convenient site of the oligonucleotide chain, being linked to P, C, N, 0 or S of the base (N), saccharide (Z), or group linked to phosphorous (X);
X is usually a pair of electrons, alkyl of from 1 to 3 carbon atoms, chalcogen (oxygen or sulfur), or amino, particularly NH;
Z is a monosaccharide, particularly of 5 to 6 carbon atom~, more particularly of 5 carbon atom.~, which may have from 0 to 1 hydroxyl groups replaced by hydrogen, and will usually be substituted by phospho-rous at the 2, 3, 5, or 6 positions, particularly at the 3 and 5 positions, and ~ubstituted at ~he one posi-tion, by the purine or pyrimidine, where the sugars mayinclude such sugars as ribose, arabino~e, xylylose glucose, galactose, deoxy, particularly 2-deoxy, derivatives thereof, etc.;
L' is a linker group which is derived from a polyvalent functional group having at least one atom, and not more than about 60 atoms other than hydrogen, usually not more than about 30 atoms other than hydro-gen, having up to about 30 carbon atoms, usually not more than about 20 carbon atoms, and up to about 10 35 heteroatoms, more usually up to about 6 heteroatoms, .particularly chalcogen, nitrogen, phosphorous, etc.;

7 1 3 1 9 1 1 q M is a moiety, particularly imparting amphi-philic properties to the compound, a hydrophobic or amphiphilic moiety which will have a ratio of carbon to heteroatom of at least about 2:1, usually at lea~t about 3:1, frequently up to greater than about 20:1, may include hydrocarbons o~' at lea~t 6 carbon atoms and not more than about 30 carbon atoms, polyoxy compounds (alkyleneoxy), where the oxygen atoms are joined by from about 2 to 10 carbon atoms, usually 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and there will be at least about 6 units and usually not more than about 200 alkyleneoxy units, more usually not more than about 100 units, generally not more than about 60 units;
Y is a bond to L or a terminal group; Y' is a bond to L', linking L' to a terminal phosphorous, or a terminal group; when a terminal group, Y and Y' are oxy, thio, amino or substituted functionalities thereof, e.g., oxyether, alkylamino, etc. or alkyl of up to about 20, usually of up to about 6 carbon atoms, Y and Y' usually not being more than about 20 carbon atoms, more usually being not more than about 10 carbon atoms;
N is any natural or unnatural base (purine or pyrimidine), capable of binding to and hybridizing with a natural purine or pyrimidine, where N may be adenine, cytidine, thymidine, guanidine, uracil, orotidine, inosine, etc.;
a is at least 4, usually at least 5, and not more than about 50, u~ually not more than about 35;
b and c are each 0 or 1.
The functional groups which find use with the linking group~, L and L', include functionalities ~uch as oxy, non-oxo-carbonyl (carboxy carbonyl), oxo-carbonyl (aldehyde or ketone), the nitrogen or 3ulfuranalogs thereof, e.g. imino, thiono, thio, amidino, etc., disulfide, amino, diazo, hydrazino, oximino, phosphate, phosphono, etc.

The linking group to the hybridizing sequence may be linked through an oxygen or sulfur present on a pyrimidine, purine, ~ugar or phosphorous group, to a carbon atom of a pyrimidine or purine, or to a phos-phorous atom. The links may be ethers to oxygen andsulfur, esters, both organic and inorganic, to oxygen and sulfur, amides, both organic and inorganic, to amines and phosphorous, and alkylamino to amino groups.
Esters include carboxylates, e.g. carboxy esters, carbamates, carbonates, etc., and phosphates, phos-phonates, etc. Of particular interest is linking at the terminal unit of the hybridizing sequence through a sugar hydroxyl, particularly at the S'-position.
The phosphorous moiety may include phosphates, phosphoramidates, phosphordiamidate, phosphorothioate, phosphorothionate, phosphorothiolate, phosphoramidothi-olate, phosphonates, phosphorimidate, and the like.
~here the phosphorous is bound to other than oxygen of the sugar, the sugar will be modified by having an amino or thio functionality at the site of binding, 90 that both amino and thio sugars may be employed to provide for novel linkages between the phosphorous and the ~ugar.
K is a chelating agent, having at least 3 heteroatoms, which are oxygen, nitrogen, or sulfur, usually combinations thereof, more usually having about 6 heteroatoms or more, which~may serve to chelate a metal ion capable of acting to inactivate, particularly to enhance cleavage, of a nucleic acid. The function-alities may include carbonyl, oxy, thiono, amino,amido, mercapto, thioether, imino, where carbonyl oxy-gens will normally be separated by at least 2 carbon atoms, usually up to 6 carbon atoms, more usually up to 4 carbon atoms; except for amido, heteroatoms will nor-mally be separated by at least 2 carbon atoms. Conven-iently, there will be at lea~t 2 non-oxo-carbonyl groups frequently at least 3 non-oxo-carbonyl group~

9 1~191 19 and not more than about 6, usually not more than about 5 non-oxo-carbonyl groups. Of particular interest are alkylene diamines and polyalkylene diamine~ having ~rom 3 to 8, usually 4 to 6 carboxyl groups, usually as carboxymethylene groups, e.g., R2N(CH2)mN(T)((CH)nN(J))X(CH2)pNR2, wherein R is a carboxyalkylene group of from 2 to 3 carbon atom~ or H, at least one R on each N being carboxyalkylene, m, n and p are the same or different and are 2 to 4, usually 2 to 3, and x is O to 2.
Illustrative chelating groups include ethy-lenediaminetetraacetic acid, dipropyleneaminepenta-acetic acid, diethylenetriaminepentaacetic acid, 2,3-bis-(2'-acetamidoethyl)succinic acid, porphyrins, phthalocyanins tetraacetic acid, and crown ethers.
A wide variety of linking groups may be employed, depending upon the nature of the terminal nucleotide, the functionality selected for, whether the linking group is present during the synthesis of the oligonucleotide, the functionality present on the hydrophobic moiety and the like. A number of linking groups are commercially available and have found exten-~ive use for linking polyfunctional compound~. The linking groups include: -OCH2CH2NHCO(CH2)nCONH-;-OCH2CH2NH-X-(CH2)nNH-;-O-P(O)(OH)NHCO(CH2)nCOHN-;-OGH2CH2NHCO~S-;-NH(CH2)nNH;-O(CH2)n ; O(CH2C 2 m ;
NH(CH2)nSYN; ~Co(cH2)nco; -SCH2CH2CO-; -CO~NYS-;
-(NCH2CH2)mCH2N-; charged and uncharged homo- and copolymers of amino acids, such as polyglycine, polyly-sine, polymethionine, etc. usually o~ about 500 to 2,000 dalton~; wherein O is phenyl; X is 2,5-quinondiyl, Y is S-(3-succindoyl) to form succinimidyl, n is usually in the range o~ 2 to 20, more u~ually 2 to 12, and m is 1 to 10, usually 1 to 6.
The amphiphilic character imparting or ~olu-bility modifying group (M) may be a wide variety of groups, being aliphatic, aromatic, alicyclic, heterocy-lo ~3191 19 clic, or combinations thereof, substituted or unsubsti-tuted, usually of at least 6, more usually at least 12 and not more than about 1000, usually not more than about 500, more usually not more than about 200 carbon atoms, having not more than about 1 heteroatom per 2 carbon atoms, being charged or uncharged, including alkyl of at least 6 carbon atoms and up to about 30 carbon atoms, usually not more than about 24 carbon atoms, fatty acids of at least about 6 carbon atom~, usually at least about 12 carbon atoms and up to about 24 carbon atoms, glycerides, where the fatty acids will generally range from about 12 to 24 carbon atoms, there being from 1 to 2 fatty acids, usually the 2 or 3 positions or both, aromatic compounds having from 1 to 4 rings, either mono- or polycyclic, fused or un~used, polyalkyleneglycols where the alkylenes are of from 2 to 8, usually of from 2 to 4 carbon atoms, more usually 2 to 3 carbon atoms, there usually being at least about 6 units more usually at least about 10 units, and usually fewer than about 500 units, more usually fewer than about 200 units, preferably fewer than about 100 units, where the alkylene glycols may be homopolymers or copolymers; alkylbenzoyl, where the alkyl group will be at least about 6 carbon atoms, usually at least about 10 carbon atoms, and not more than about 20 carbon atoms; alkyl phosphates or phosphonates, where the alkyl group will be at least 6 carbon atoms, usu-ally at least about 12 carbon atoms and not more than about 24 carbon atoms, usually not more than about 20 carbon atoms, or the like.
The "M" group may be charged or uncharged, preferably being uncharged. Illustrative groups include polyethylene glycol having from about 40 to S0 units, copolymers of ethylene and propylene glycol, laurate esters of polyethylene glycols, triphenyl-methyl, naphthylphenylmethyl, palmitate, distearyl-glyceride didodecylphosphatidyl, cholesteryl, arachi-donyl, octadecanyloxy, tetradecylthio, etc.

Functionalities which may be present include oxy, thio, carbonyl, (oxo or non-oxo), cyano, halo, nitro, aliphatic unsakuration, etc.
In designing the nucleic acid sequence, it will be desirable to have a high affinity between the subject composition and the target single stranded nucleic acid sequence. Sequences will preferably be selected having greater than 40~ GC content, more preferably greater than 50% and may have 60% or more GC
content. For optional selectivity, the melting temper-ature of the hybrid to be formed should be 5 to 10C
above the ambient temperature at which the hybrid forms, usually the ambient temperature being 37C in a mammalian host. For mammalian hosts, the melting temperature will generally be chosen to be about ~2-50C. The target sequence should be selected to be relatively free of ~econdary and tertiary ~tructure.
In many mRNA's, an open region will be present in the vicinity of the start codon (AUG).
In preparing the subject compositions, various strategies may be employed, depending upon whether "M"
is present, the nature of "M", the nature of the oligo-nucleotide and the nature of the linking group. Thus, so long as care is taken that the addition of the two different groups, '7M" and the chelating group, do not interfere with one another, the groups may be added sequentially.
One technique for providing the chelating agent may be found in Dryer and Dervan, supra. In thi~
technique, a modified nucleoside is employed during the synthesis of the oligonucleoti~e. Thymidine may be modified at the methyl group by providing for a carboxy alkyl group. The carboxy group may then be further functionalized with an alkylene diamine, and the amino group employed for amide formation with a carboxy con-taining chelating agent. The modified thymidine may then be employed as a nucleotide reagent in the auto-mated synthe~i~ of the oligonucleotide.

12 l~ql 19 Alternatively, the final nucleotide adduct in the synthesis of the oligonucleotide may be functional-ized in a variety of ways which may serve to act as a linking unit to the chelating agent. For example, 5 after removal of the trityl protective group an amino-ethanolphosphoramidite is addecl, as described by the supplier (Applied 8iosystems, Foster City, CA) to pro-vide for an available amino group. After deblocking and removing the oligonucleotide chain from the sup-10 port, the amino group is then available for linking tothe chelating agent. Alternatively, the oligonucleo-tide i9 phosphorylated employing a polynucleotide kinase, followed by formation of a phosphoramidate using an activating agent, such as 1-methylimidazole or a water soluble carbodiimide, in the presence of an alkylene diamine, providing for an amino functionality (Chu and Orgel, DNA (1985) 4:327-331). A further alternative is to deblock the oligonucleotide while retaining the oligonucleotide on the support, followed 20 by treatment with carbonyldiimidazole. After removal of excess of the carbonyldiimidazole, a diamine may be added to provide an aminoalkylcarbamate (Wachter et al., Nucl. Acids ~es. (1986) 14:7985-7994~.
Where "M" is to be added, a mercaptan group 25 may be provided a~ part of the functionalizing agent or separate from the f`unctionalizing agent. The mercaptan group may be pa~rt of the linker to the support or may be part of the functionalizing agent of the oligo-nucleotide, where both the chelating agent and "M" may 3o be bound to the same linking group. Besides mercaptan groups, maleimido groups may be employed, where "M" or the chelating agent may have a mercaptan group to form a thioether.
Various active functionalities can be employed 35 to produce a covalent linkage, such a~ isocyanate~, isothiocyanates, diazo groups 3 imino chlorides, imino e~ters, anhydride~, acylhalide~, sulfinylhalides, 13 131~1 19 sulfonyl chlorides, etc. Conditions for carrying out the various reactions and joining non-nucleotide moieties to nucleotide moieties may be found in Chu and Orgel DNA (1985) 4:327-331; Smith et al. Nucl. Acids 5 Res. (1985) 13:2399-2412.
The linking arms, "Mt', and the chelating moiety may be added at various times, depending upon the particular reaction scheme. For the most part, the chelating agent may be part of a nucleoside and be included in the synthesis of the oligonucleotide or may be added after oligonucleotide formation. "Ml' will normally be added after oligonucleotide formation.
For the most part, reaction conditions will be mild and will employ polar solvents or combinations of polar and nonpolar solvents. Solvents will vary and include water, acetonitrile, dimethylformamide, diethyl ether, methylene chloride, dimethylsulfoxide, etc.
Reaction conditions will be for the most part in the range of about -100-60C. Usually, after completion of the reaction between components of the conjugate, the resulting product will be subjected to purification.
The manner of purification may vary, depending upon whether the oligonucleotide is bound to a support.
For example, where the oligonucleotide is bound to a support, after addition of the linking arm to the oligonucleotide ! unreacted chains may be degraded, so as to prevent their contaminating the re~ulting product. Where the oligonucleotide is no longer bound to the support, whether only reacted with the linking arm or as the conjugate to the chelating agent or as the final product, each of the intermediates or final product may be purified by conventional techniques, such as electrophoresiq, solvent extraction, HPLC, chromatography, or the like. The purified product is then ready f'or use.

The subject products will be selected to have an oligonucleotLde sequence complementary to a sequence of interest. The sequence of interest may be present in a prokaryotic or eukaryotic cell, a virus, a normal or neoplastic cell. The sequences may be bacterial sequences, plasmid sequences, viral sequences, chromo-somal sequences, mitochondrial sequences, plastid sequences, etc. The sequences may involve open reading frames for coding proteins, ribosomal RNA, snRNA, hnRNA, introns, untranslated 5'- and 3'-sequences flanking open reading frames, etc. The subject sequences may therefore be involved in inhibiting the availability of an RNA transcript, inhibiting expres-sion of a particular protein, enhancing the expression of a particular protein by inhibiting the expression of a repressor, reducing prol~feration of viruses or neo-plastic cells, etc.
The subject conjugates may be used in culture or in vi~o for modifying the phenotype of cells, limit-ing the proliferation of pathogens such as viruses,bacteria, protista, mycoplasma, or the like, or induc-ing morbidity in neoplastic cells or specific classes of normal cells. Thus, one can use the subject composi-tions in therapy, by administering to a host subject in a diseased state, one or more of the subject composi-tions to inhibit the transcription and/or expression of ; the n?tive genes of a cell. The subject compo~itions may be used for protection of a mammalian host from a variety of pathogens, e.g., enterotoxigenic bacteria, Pneumococcus, Neisseria, etc.; protists, such as Giardia, Entamaeba, etc.; neoplastic cells, such as lymphoma, leukemia, carcinoma, sarcoma etc.; specific B-cells, specific T-cells, such as helper cells, supressor cells, CTL, NK, etc.
; 35 The ~ubject compositions will be selected so as to be capable of inactivating sequencas of interest, particularly mRNA, or in some circumstances the subject composition can be used with other nucleic acid moieties, e.g., tRNA, snRNA, DNA, e.g., plasmids, viru~es, etc. Thu~, the subject compositions may bind to mRNA and provide for cleavage of the mRNA, so as to prevent the expression of a product. By employing sequences which are relatively inert to degradation, the lifetime of the chelate conjugate may be substan-tially extended in the host cell, so as to have a rela-tively high kill ratio per sequence.
The subject se4uences may be complementary to such sequences as sequences expressing gro~th factors, lymphokines, immunoglobulins, T-cell receptor sites, MHC antigens, DNA or RNA polymerases, antibiotic resis-tance, multiple drug resistance (mdr), gene~ involved with metabolic processes, in the formation of amino acids, nucleic acids, or the like, DHFR, etc. as well as introns or flanking sequences associated with the open reading frames.
The subject composition~ may be administered to a host in a wide variety of ways, depending upon whether the compositions are used in vitro or in vivo.
In vitro, the compositions may be introduced into the nutrient medium, so a~ to modulate expression of a par-ticular gene by transferring across the membrane into the cell interior such as the cytoplasm and nucleus.
The ~ubject compo~itions may find particular use in ; protecting mammalian cells in culture from mycopla~ma, for modifying phenotype for research purposes, ~or evaluating the effect of variation of expression on 3~ various metabolic processes, e.g., production of parti-cular products, variation in product distribution, or the like. While no particular additives are necessary for tran~port of the subject compositions intracellu-larly, the subject compositions may be modified by being encapsulated in liposomes or other vesicle, may be used in conjunction with permeabilizing agents, e.g., non-ionic detergents, Sendai virus, etc~

'``4~
16 ~31~
For in vlvo administratlon, depending upon lts -particular purpose, the sub~ect compos~tlons may be admlnlstered in a varlety of ways, such as in~ectlon, infu~ion, tablet, etc., ~o that the composltions may be taken orally, parenterally, intravascularly, intraperi-toneally, subcutaneou~ly, intrale310nally, or the llke. The compositions may be ~ormulated in a variety of ways, being di~persed in variou~ phy~iologically acceptable media, such as delonized water, water, phos-phate buffered saline, ethanol, aqueous ethanol, formu-lated ln the lumen of vesicles, microencap~ulated, etc.
Becau~e of the wide variety of applications and manners of admlni~tration, no particular composi-tion can be suggested. Rather, as to each indication, the subject compositions may be tested in conventional ways and the appropriate concentration~ determined empirically. Other additive3 may be included, such as ~tabilizers, buffer~, additional drug~, detergents, etc. The~e additives are conventional, and would generally be present in le~s than about 5 wt~, u~ually les~ than 1 wtS, being pre~ent in an effective do~age, as appropriate. For filler3 or excipient~, these may be as high a~ 99.9S of the composition, depending upon the amount of active materlal necessary.
The following example3 are presented by way of illustration not by way of limitation.

Synthe~ls of Diethylenetriamine pentacetic acid (DTPA) Con~u~ated Oligomer~
Chemical Synthesls of DNA. The chemical ~yn-thesi~ of DNA was carried out using 311ght modifica-tion~ of the conventional phosphoramidite methods on an Applied Biosystems tModel 381) DNA synthesizer. The method used i~ a modlfication of the technique described by Caruthers and coworkers (Beaucage and Carutherq, 1984, European Patent Application 61746 issued March 20, 1985.
~'`'' '~
., , In this technique, nucleoside phosphoramidites dissolved in anydrous acetonitrile are mixed with tetrazole and sequentially coupled to the 5' hydroxy terminal nucleotide of the growing DNA chain bound to controlled pore glass (CPG) support~ via a succinate spacer (Matteucci and Caruthers. Tetrahedron Letters (1980) 21:719-722). Nucleoside addition is followed by capping of unreacted 5' hydroxyls with acetic anhydride, iodine oxidation, and 5' detritylation in trichloroacetic acid-methylene chloride. The resin bound oligomer i~ then dried by extensive washing in anhydrous acetonitrile and the process repeated.
Normal cycle times using this procedure are 12 minutes with condensation efficiencies of >98~ (as judged by trityl release).
Chemical Synthesis of Amine Linker Arm Containing DNA Oli~onucleotides Bound to CPG Glass Beads. The completed fully blocked DNA chains can subsequently be modified to contain a 5' amino linker arm while ~till attached to the CPG support. Several method~ have been used to accomplish this.
Aminolink Procedure for 5' Amine Linker Arm Attachment. At the end of the synthesis, trityl is removed from the product oligonucleotide chains and an aminoethanolphosphoramidite is added to the 5' hydroxyl u~ing the Aminolink procedure developed and marketed by - Applied Biosy~tems (Foster City, CA). The resin bound oligonucleotide i~ then deblocked and released from the column using a method appropriate to the type of phos-phate linkage prasent. For normal phosphodie~ter~, hydrolysis in concentrated ammonia is appropriate. For DNA triester~ and methylpho~phonateq, ethylene diamine (EDA) phenol deblocking followed by ammonia or EDA:
ethanol release is appropriate. Result~ indicate that all of the cyanoethyl-phosphorus and aryl-amide base blocking groups are removed under these condition~.

Addltion of_Amine Containlng Llnker Arms U~ing Phosphoramldate Linkage. One alternative is the use of the technique of Chu and Orgel, Proc. Natl. Acad. Scl.
USA (1986) 82:963-967 to add linker arms to the 5' end of any ollgonucleotide. In thls instance, the oligo-nucleotide is first phosphorylated u~ing polynucleotide kina~e. After purification by polyacrylamide gel electrophoresis, the product DNA containing a free 5'-hydroxyl i~ phosphorylated with the forward reaction of T4 polynucleotide kinase according to Chu and Orgel, ~upra (1986). Pho~phorylated oligomer~ are ~eparated from unreacted ATP using a C-18 reverse phase column (Waters SEP-PAK) according to the direction of the manufacturer. The phosphorylated oligomer i3 then treated with 0.1M 1-methyl imidazole, 0.1M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 0.1M alkyl diamine (e.g., hexane diamine), pH 7, under conven-tional conditions in an aqueous medium to form the desired phosphoramidate containing a free amine with the following structure oligomer-P-NH-(CH2)n-NH2 ~H
Addition of An Amine Linker Arm Usin3 Carbonyl Diimidazole (CDI). A third alternati~e to the addition of an amine linker arm to the 5' end of any oli~o-nucleotide is to use carbonyl-bis (imidazole). In this technique, the CPC bound, ba3e blocked oligomer (trityl of~) is fir~t treated with CDI (50 mg/ml) in dry aceto-ni~rile for 30 to 45 minute~ to form the imidazole carboxylic acid ester. The exce~s CDI i~ then wa~hed off with acetonitrile and water and the diamine of choice added to the column generally in a mixture of acetonitrile or dioxane and water~ The dlamine form~ a * Trademark J
~ .
" ~ ., ~

1319~ 1~

stable carbamata linka~e after a brief incubation. The oligomer can then be deblocked and relea~ed ~rom the column under conditions approprLate to the type of pho~phate llnkage present.
Attachment o~ the Clea~age Unlt DTPA. Once the amine terminated oligomer i9 deblocked and charac-terlzed, the cleavage unit was added using the ~ollow-ing method. Ten units of the o:Ligomer were di~ol~ed in 100 ~1 dimethylformamide containing 0.1 M DTPA (bi~-anhydride). The mixture was incubated ~or 1-2 hours at room temperature, and the exces3 DTPA removed by gel filtration and concentration on Centricon C1U mem-branes. The final product was dried in ~acuo and dis-solved to a final concentration of 1 mM in water (ba~ed on its optical den~ity at 260nM) and stored frozen.
Thi~ solution was ~table ~or at least 1 month. The compound was homogeneous as judged by polyacrylamide gel electrophoresi3.

Synthe~is o~ DTPA Derivative-~ of Normal DNAs U~ing Imidazole Activated Carboxylic Acid Esters and Lon~ Chain Aminoalkanes In thi~ example, a 20 nucleotide DNA comple-mentAry to the initiation region of mouse bata globin mRNA was synthe~ized according to the method giYen in EXAMPL~ 1. After ~ynthe~is, the product material wa~
retained on the synthe~is support wlth trityl removed from the 5' end of the molecule. The solid material wa~ then thoroughly wa~hed with anhydrou~ acetonitrile and blown dry under a stream of dry argon. U~ing a plastic syringe, 1 cc of 0.3M carbonyldiimidazole di~-solved in anhydrou~ acetonitrile wa~ pu3hed slowly through the synthe~is column containing the support bound ollgomer o~er a period of 45 minutes. The 5' carbonyllmidazole acti~ated oligomer on the column wa~
then washed free o~ exce3s reagent with 15 ~1 of aceto-*Trademark nitrile and then treated with 0.2 M decanediamlne lnacetonltrlle:water (10:1) for 30 minutes.
The material on the column was washed free o~
unreacted decane-diamine with acetonitrlle and water, and then eluted from the column ln concentrated ammon-ium hydroxlde. After removal from the column, the ammonium hydroxide ~olution containing the oligomer conjugate was placed in a sealed Yial and incubated 5 hour~ at 55C.
The product iY then lyophilized several tlmes from 50% aqueous ethanol and purified via reversed phase HPLC C-8 silica columns eluting 5 to 50S aceto-nitrilet25 mM ammonium acetate, pH 6.8 ln a llnear gradient. If required, the material was further puri-fied by ion-exchange HPLC on Nucleogen DEAE 60-7 elut-ing 20~ acetonitrile/25 mM ammonium acetate, pH 6.5.
The recovered product i9 then characterized by gel electrophoresis in 15S polyacrylamide gel~ carried out a~ descrlbed by Maxam and Gilbert (Meth Enzymol.
~1980) 68:499-560). Oligonucleotides in finished gels are visualized using Stains-all.
As a further check on the material, the pre~ence of a primary amine can be determined by two methods. Flrst, reactlon with fluore camine produced a fluorescent product characteri~tic of the presence o~ a primary amine while no rluorescence i9 observed with ~imilarly treated control oligomers o~ the ~ame type~, but lacking the amine linker. Second, the decane con-~ugate was dissolved in 100 ~l 0.1 M ~odium bicarbonate to which was added 1 mg of fluorescein isothiocyanate.
After 1 hour of incubation, the unreacted FITC was removed by gel filtration chromatography on Sephadex G-25 spun columns. The product was then analyzed by polyacryla~ide gel electrophore~l~ as described above, and the fluorescent band product vlsualized under UV
illumination. A single fluorescent band is observed which corresponds to the ol~gomer ~isualized by subse-quent staining with Stains-all.
*Trademarks ,is,~,j . . ~

The product of this reaction is an aminoalkyl carbamate coupled to the 5' end of the oligonucleotide.
The alkylcarbamate i~ stable to moderate exposure to concentrated ba~e. The free amino group distal to the carbamate linkage is available for subsequent deriva-tion which can be accomplished according to the method given in EXAMPLE 1. The structure of the final con-jugate synthesized by this method is illustrated as:

oligomer - 0 - C - NH - (CH2)10 - NH - C - DTPA

Synthesis of DTPA Derivatives of DNAs Using Imidazole Activated Carboxylic Acid_Esters and a Poly-D-lysine Linker In this example, a 25 nucleotide DNA comple-mentary to the initiation region of rabbit beta globin mRNA was synthesized according to the method given in EXAMPLE 1. After synthesis, the CPG support containing the oligomer was treated with 80% acetic acid for 30 minutes to remove trityl from the 5' end of the mole-cule. The solid material was then thoroughly washed with anhydrous acetonitrile and blown dry under a ~tream of dry argon and treated with 0.3M CDI as in EXAMPLE 4. The 5' CDI activated oligomer on the column waq then washed free of exces~ reagent with 15 ml of acetonitrile and then treated with 0.2 M poly-D-lysine (MW+1,000) dissolved in 50% acetonitrile containing 3 0.1 M sodium phosphate, pH 8 for 16 hours at room temperature.
The material on the column was washed free of ~alts and unreacted polyly~ine with water and aceto-nitrile and then eluted from the column in concentrated ammonium hydroxide. A~ter removal ~rom the column, the ammonium hydroxide solution containing the oligomer 22 1 Sl qll 9 con~ugate wa~ incubated for 5 hours at 55C in a sealed glas~ vial. The product was then lyophilized 3everal times from 50~ aqueous ethanol and purifled Yia gel filtration chromatography on TSK G4000SW in 100 mM Tri~
buf~er, pH 7.5. The presence of a primary amlne was determined by reaction with fluorescamine. No fluores-cence was observed with control oligomers lacking the polyamine linker.
The polyamine conjungate cannot ea~ily be characteri2ed by gel electrophoresis since it is elec-trostatically and molecularly polydi~perse. In order to render the polyamine conjugate negatively charged, the complex was reacted with FITC to label the molecule and to neutralize the positive charges on the amines.
This wag accomplished by dissolving a portion of the material in 100 ~1 0.1 M sodium bicarbonate to which was added 1 mg of fluoresceinisothiocyanate. After 1 hour of incubation, the unreacted FITC was removed by gel filtration chromatography on SephadeX*G-25 spun 20 columns (Maniatis et al., Molecular Cloning - A
Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, New York (1982)). The product was then analyzed by polyacrylamide gel electrophoresis carried out as described by Maxam and Gilbert (Meth. Enzymol.
(1980) 60:499-560) and the fluorescent band product ~isualized under UV illumination. A ~ingle broad ~luore~cent band i9 ob3erved which corre3ponds~to the DNA visualized by Stains-all.
The struoture of this con~ugate may be illu9trated as having the general formula:

O O O
n n n oligomer - O - C - NH - (CH - C - NH)n - C-CH-COOH
(1H2)4 (1H2)4 ~H 1H2 O ~ ~ - DTPA

*Trademark ... ,~, ~,.,j By varying the reaction excess of the DTPA or the molecular size of polylysine used, it is possible to construct conjugate~ with varying degrees of substi-5 tution, si~e and charge. The ability to vary theseproperties of the complex make it po~sible to design the use of the compound in various applications.

Synthesis of DTPA Derivatives of DNA Methylphosphonate~
The chemical synthesis of DNA methylphophon-ates may be carried out using a modification of the phosphochloridite method of Letsinger (Letsinger et al., J. Am. Chem. Soc. (1975) 97 :3278; Letsinger and Lunsford, J. Am. Chem. Soc. (1976) 98 :3655-3661; Tanaka and Letsinger, Nucl. Acids Res. (1982) 1~:3249).
However, the preferred method and the one used in this example uses methyl phosphonamidites (Applied Biosy3tems, Foster City, CA). The method for performing the synthesis uses exactly the ~ame 3teps and reaction times a3 in conventional DNA synthesis, with the exception that THF rather than acetonitrile is used to dissolve the phosphonamidites due to their 25 reduced solubility in the latter. Normal cycle times using thi~ procedure are 15 minute3 with condensation efficiencies of >94~ (as judged by trityl release).
The ultimate base may be added as the cyanoethyl pho3-photriester which yields, upon cleavage in base, a 5 ' : 30 terminal pho~phodiester. Thi3 step make~ it po3sible to radiolabel the oligonucleotide, purify and sequence the product using gel electrophoresis at intermediate stages of preparation (Narang et al, Can. J. 8iochem.
(1975) 53:342-394; Miller et al., Nucl. Acids Res.
.... ~ 35 (1983) 11 :6225-6242).
An amine terminated linker arm i3 then added a~ follows. Trityl i~ removed as before and the resin 24 131~1 19 treated with 0.2 M Aminolin~ (Applied Bio~y tems, Foster City. CA) di~solved in dry acetonitrlle contain~
ing 0.2M dimethylaminopyrodine for 5 minute~. The linker arm oligonucleotide is then oxidized in iodine and wa~hed in acetonitrile as above. Capping with acetic anhydride i~ not performed ~ince any deblocked primary amine would be modified to the base stable acetamide and thu~ unavailable for further reaction.
At the end of the synthe~is, the a~ine termi-nated linker arm methylpho~phonate oligomer i~ ba~edeblocked a~ follow~. The resin containing the DNA i~
removed from the column and placed in a water jacketed column and incubated in 1-2 ml phenol:ethylene diamine (4:1) for 10 hours at 40C. At the end Or the incuba-1~ tion in phenol:ethylene diamine, the re~in i~ washedfree of the phenol reagent and released ba e protecting groups using methanol, water, methanol and methylene chloride in ~ucce~slon. After drying in a stream of nitrogen, the intact, basa-deblocked chains are cleaved from the ~upport u~ing EDA:ethanol (1:1) or brier treatment with room temperature ammonium hydroxide.
PuriYication of the amine terminated DNA
methylphosphonate i~ then performed aY ~ollows. The material is ~irst lyophilized several times ~rom 50S
aqueous ethanol and purified via re~ersed pha~e HPLC C-8 311ica column3 eluting 5 to 50% acetonitrile/25 mM
ammonium acetate, pH 6.8 in a linear gradiant. Amine containing ~raction~ a~ determined by fluore~camine reactivity are pooled and the product recovered by 3o drying in vacuo and ~urther puriSied by ion-exchange HPLC on Nucleogen DEAE 60-7 eluting 20% aceonitrile/25 mM ammonium acetate, pH 6.5.
The purlfied product i~ then con~erted to the DTPA derivative as in EXAMPLE 1. Purification o~ the complex is then e~rected a~ preriously described.
Alternativelyt unbound ollgonucleotide 19 remoYed by gel filtration on Sephadex*G-I00 or HPCFC*on TSK
G4000S~ alut i ng 10 mM Tris, pH 7.5.
,~'! * Trade mark ,~
~ .

2s 13191 19 The structure o~ the final product of this procedure is illustrated as:

O O
,. ..
5 oligomer - P - O - (CH2)2 - NH - C - DTPA
MP
OH

Synthesis of DTPA DerivatiYes of DNA
Ethyltriesters Using the Phosphoramidite Approach The synthesis of the title compound triesters is performed according to the method of Zon and cowork-ers (Gallo et al., Nucl. Acids Res. (1986) 14:7405;
Summers et al., Nucl. Acids Res. (1986) 14:7421-7436).
The method of synthesis is similar to that which i~
used for in situ production with ethyl triesters as described by Letsinger (Letsinger et al., J. Am. Chem.
Soc. (1975) 97:3278; Letsinger and Lunsford, J. ~m.
-20 Chem. Soc. (1976) 98:3655-3661; Tanaka and Letsinger, Nucl. Acids Res. (1982) 10:3249). In brief, fully blocked dimethoxytrityl nucleosides are derived by repeated lyophilization from benzene, dis~olved in anhydrous acetonitrile/2,6-lutidine (8:2) and added 25 dropwi3e to a stirred solution of chloro diisopropyl-amino ethoxyphosphine in the same solvent at -70C.
The product is recovered by aqueou~ extraction, in vacuo drying and silica gel chromatography.
- The chemical synthesi~ of DNA can be carried 30 out using slight modifications of the conventional phosphoramidite methods. In this technique, nucleoside phosphoramidites di3solved in anhydrous acetonitrile are mixed with tetrazole and sequentially coupled to the 5' hydroxy terminal nucleoside bound to CPG.
35 Nucleoside additinn i~ followed by capping of unreacted 5' hydroxyl~ with acetic anhydride, iodine oxidation, and 5~ detritylation in trichloroacetic acid-methylene chloride. The resin bound oligomer is then dried by extensive washing in anhydrou~ acetonitrile and the process repeated. Normal cycle time~ u~ing thi~ proce-dure are 17 minute~ with condensation efficiencie~ o~
>96~ (as ~udged by trityl release). The terminal re~i-due is conventionally added a~ a diester in order to facilitate radiolabeling and purification. The 5' terminal trityl group is left if HPLC purification is desired, but generally the 5' terminal trityl i~
removed and the aminolink procedure described in EXAMPLE 1 is used.
At the end of the synthesi~, the fully blocked product i~ ba~e-deblocked as follow~. The re~in containing the fully protected DNA is removed from the column and placed in a water ~acketed chromatography column. The re~in is then incubated in 1-2 ml phenol-ethylene diamine (4:1) for 10 hours at 40C. At the end of the incubation in phenol:ethylene diamlne, the resin is washed free of the phenol reagent and relea~ed base protecting groups using methanol, water, met~anol and methylene chloride. After drying in a stream of nitrogen, the intact, base^deblocked chain~
are cleaved from the support u3ing EDA:ethanol (1:1) or brie~ treatment with room temperature ammonium hydroxide.
Purification of the aminolink DNA ethyl-triester product 19 ~hen performed a~ follow~. The material i3 ~ir~t lyophilized several times from 50S
aqueous ethanol and purified via reversed pha~e HPLC C-30 8 silica columns eluting 5 to 50S acetonitrile/25 mM
ammonium acetate, pH 6.8 in a linear gradient. Amine containing fraction~ a~ determined by fluorescamine reactivity are pooled and the product recovered by drying in vacuo and further purified by ion-exchange 35 HPLC on Nucleogen DEAE 60-7 eluting 20% acetonitrlle/25 mM ammonium acetate, pH 6.5.
*Trademark ~.
'`' ,~ f~);

1 3 1 q 1 1 q The product olLgonucleotide i9 then suitable for coupling to DTPA and purification by the techniques previously described.
The structure of the final product of this procedure is illustrated as:

O O
,. ..
oligomer - P - O - (CH2)2 - NH - C - DTPA
ETE
OH

Synthesis of DTPA Derivatives of DNA Alkyl and Aryltriesters Using the Phosphate Triester Approach A preferred method for the production of the oligonucleotide triesters of variable alkane chain length is via conventional phosphate triester chemistry to synthesize the desired sequences as the p-chlorophenyl phosphate triesters. Upon completion oftha synthesis, the fully protected oligonucleotide chlorophenyltriesters bound to the synthesis support are subjected to ester exchange in the presence of tetrabutylammonium fluoride and the desired alcohol.
This basic method for the construction of DNA oligo-nucleotides is classical DNA synthesis chemistry and presents no problems. The essential chemistry i~ well described (Gait, Oligonucleotide Synthesis: A Practical Approach IRL Press, Washington, D.C. (1984)) and can be used with little modification. An alternative phos-phite based chemistry which is much more rapid and gives equivalent yields is set forth below.
The chemical synthesis of DNA p- or o-chlorophenyl phosphotriestars was carried out using a modification of the phosphochloridite method of Letsinger (Letsinger et al., J. Am. Chem. Soc. (1975) ; 97:3278; Letsinger and Lunsford, J. Am. Chem. Soc.

(1976) 98:3655-3661; Tanaka and Letsinger, Nucl. Acids Res. (1982) _:3249). A programmable, automated DNA
synthesizer used for pho~phomonochloridite based syntheses (Alvarado-Urbina et al., Science (1981) 21 4:270-273.
Fully blocked and carefully dried nucleosides dissolved in anhydrous acetonitrile, 2,6-lutidine and activated in situ with chlorophenoxydichlorophosphine are sequentially added to the 5' hydroxy terminal 10 nucleotide of the growing DNA chain bound to controlled pore glass supports via a succinate spacer (Matteucci and Caruthers, Tetrahedron Lett. (1980) 21 :719-722).
Derivatized glass supports, fully block nucleosides and other synthesis reagents are commercially available 15 through Applied Biosystems (San Francisco, CA) or American Bionuclear (Emeryville, CA). Nucleoside addition is followed by capping of unreacted 5' hydroxyls with acetic anhydride, iodine oxidation, and 5' detritylation in trichloroacetic acid-methylene 20 chloride.
The resin bound oligomer chlorophenyltriester is then dried by extensive washing in anhydrous aceto-nitrile and the process repeated. Normal cycle times using this procedure are 13 minutes with condensation 25 efficiencies of >92% (as judged by trityl release).
The ultimate base may be added as a methyl phospho-triester which yields, upon cleavage in base, a 5' terminal phosphodiester. This step makes it po~sible to radiolabel the oligonucleotide and to purify and 30 3equence the product using gel electrophoresis (Narang et al., Can. J. Biochem. (1975) 53:392-394; Miller et al., Nucl. Acids Res. (1983) 1 :6225-6242).
The fully blocked material bound to the synthesis qupport is then subjected to ester exchange 35 in the presence of tetrabutylammonium fluoride (TBA~) and the desired alcohol. This method yields rapid and quantitative alcohol exchange. The reaction is com-plete within 20 minutes for most aryl and alkyl alco-hols which are capable of forming stable products. With any alcohol, the presence of trace amounts of water can affect the overall yield. Thus, care must be taken to 5 used anhydrous alcohols at this step.
In this example, anhydrous n-propanol is used to dissolve TBAF to a final concentration of 0.2 M.
The solution is then percolated slowly over the resin containing the oligomer chlorophenyl triester and 10 allowed to react for about 1 hour at room temperature.
The resin is then washed with methanol and acetonitrile and dried under a stream of dry argon. Amine linker arm addition, deblocking and purification are then effected as in EXAMPLE 2. DTPA conjugation is then 15 performed as in EXAMPLE 1. The final yield of conju-gate is about 10% of the starting equivalents of nucleoside resin used.
The structure of the final product is illu-strated as:
O O
,~ ,.
oligomer - C - NH - (CH2)10 ~ NH - C - DTPA
PTE
OH

Effect of DTPA Conjugate~ on the Synthesis of Hemoglobin in Mouse MEL Cells The effectiveness of oligomer DTPA con~ugate 30 mediated HART (Paterson et al., Proc. Natl. Acad. Sci.
USA (1977) 74:4370; Haqtie and Held, Proc. Natl. Acad.
Sci. USA (1978) 75:1217-1221) was determined in cultured cells incubated in the presence or absence of the oligomer. The cells used were Friend murine 35 erythroleukemia (M~L) cells which can be induced to synthesize hemoglobin by a variety of agentq including DMSO and butyric acid (cf. Gusella and Houseman, Cell 1~191 lq (1976) 8:263-269). Friend leukemia cells were grown in culture using well known techniques in a C02 incubator.
Hemoglobin synthesis was induced using 1.5% DMSO.
Induced cells expressing hemoglobin were 5 visualized by benzidine treatment which stains globin producing cells blue (Leder et al., Science (1975) 190:893). Cells were exposed to selected oligonucleo-tides and DTPA conjugate at concentrations ranging from 0.1 ~IM to 50 IIM during a 11- to 5-day induction period.
10 Controls included mock-treated cells and cells treated with unmodified oligomers of the same sequence.
Treated cells were scored for globin production based on staining intensity and the results compared to controls. Cell death or damage due to treatment was 5 scored by Trypan blue exclusion.
The following Table (Table I) indicates the speciIic sequences synthesized.

TABLE I
DNA SEQUENCES SYNTHESIZED AND CO~JUGATED
FOR USE IN ÆLL CULTURE EXPERIMENTS
Probes Synthe~ized Antisense to Mouse Beta-globin mRNA ,~ GC Sequence (3' to 5') _ MBG 15 methylphosphonate 60% g tac cac gtg gac tG
MBG 15 methylphosphonate-C2amine 60% g tac cac gtg gac tGp-O-(CH2)2-NH2 ~G 15 methylphosphonate 60% g tac cac gtg gac ~Gp-~(CH2)2 DTPA conjugate -NH-C(O)-DTPA
MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA C
MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA
- C2-amine Cp O (CH2)2 NH2 MBG 20 antisense 55% G TAC CAC GTG GAC TGA CTA
C6-amine C-O-(CO)-NH-(CH2)6-NH2 MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA
C~,-amine DTPA Cp-O-(CH2)2-NH-C(O)-DTPA
conjuga~e ., _ 13191 lq a) Lower case letters represent nucleosldes coupled to the 3' adjacent nucleoside via a methylphosphonate linkage. Upper case letters represent 3' adjacent normal phosphodiester 1inkage. C
derivatives are formed from the conden~ation of ethanolamine wi~h a 5' terminal phosphate via an ester linkage. C6 and C10 derivatives are the corresponding diamines coupled via an alkyl carbamate linkage to the 5' terminal hydroxyl. DTPA represents diethylenetriamine pentaacetic acid.

The data in the ~ollowing Table (Table II) show that DTPA conjugated oligomers were approximately 500 times more effective than control oligomers with or without the amine linker attached. Significant cyto-toxicity was observed only at concentrations above 10 ~M, about 100 times the minimum dose for a significant effect.

3o TABLE II
The Effect of Cleaver Conjugate Oligonucleotides in Preventing the Synthesis of Hemoglobin in Cultured Cells _ _ _ % Inhibition Treatment (1) Conc. ~ Viable Cells Clobin+ Cells Experiment #1 - ~ffect Compared to Other Constructs.
DMSO Control 46% 0%
10 MBG-20 Antisense 50 ~M 50% 41~
MBG-20-C2amine 50 ~M 61% 41%
MBG-20-DTPA 50 ~M 0% 100%
Conjugate Experiment #2 - Concentration Dependence of DTPA Conjugate DMSO Control 1.5% 65% 0%
Solvent Control n.d 0%
EDTA Control 1 ~M 65% 0%
MBG-20-DTPA 1 ~M 50% 98%
Conjugate 57% 96%
500 nM 34% 95%
14% 96%
300 nM 65% 84%
65% 84%
200 nM 78% 58%
100 nM 69% 56g 76% 60%
. - 10 nm 67% 47%
74% 61%
1 nM 70~ 41%
3 0.1 nM 72% 2%
none nd 0%

Table II
(continued) Experlment t3 - E~fect o~ ~ethylphosphonate DTPA Con~ugate 5 Treatment (1) Conc. '~ Viable Cell~ S Inhibitlon DMS0 Control 50~ 0%
MBG 15 methyl- 100 nM 50~ 48%
pho~phonate 10 nM 51~ 43%
DTPA Con~ugate 1 nM 54% 3f%
0.1 nM 54% 37%

(1) C2 derivative~ are formed from the condensation o~
ethanolamine with a 5' terminal phsophate Yia an e~ter linkage. DTPA repre~ent~ diethylenetriamine pentaacetic acid.

It is evident from the above result~, that conjugate~ o~ a chelating agent and an oligonucleotide ~equence mar be used to preferentially inhibit the - 20 expre~sion o~ a sequence in a viable cell. In this manner, cell~ can be modified in a variety of ways, ~o a~ to change the phenotype or to ~electively kill cells. The con~ugate i9 ~table and does not require that a metal be non-covalently bound to the chelating agent prior to use in order to achieve ef~ectivene~s. In addition, the subJect compo~itions can be used in vi~o or in vitro, allowing ~or selection of cells, enhancing actiYity of particular cells, reducing actlvity o~ particular cells, or permitting ~elec-tion Or a particular clas of cells. A Yariety of con~u-3 ~ates can be produced, which will ha~e long hal~ live~, ~oa3 to be able to pro~ide for destruction of a large number Or RNA sequences for each molecule con~ugate.

Although the foregoing invention has been deæcribed in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (28)

1. A method of inhibiting the utilization of RNA in a cellular process in vitro, said method comprising:
contacting cells in vitro with a composition capable of entering contacted cells, said composition comprising an oligonucleotide chelate conjugate capable of hybridiz-ing to an intracellular RNA sequence, said oligonucleotide chelate conjugate comprising an oligonucleotide conjugated to a metal-ion free chelating agent capable of chelating a metal ion which is capable of initiating a reaction that results in the inactivation of said RNA sequence.

2. A method according to Claim 1, wherein said oligonucleotide is at least about 50% G + C.

3. A method according to Claim 1, wherein said chelating agent is a polyamino-polycarboxylic acid chelating agent.

4. A method according to Claim 3, wherein said chelating agent is an alkylenepolyamine polycarboxymethylene chelating agent.

5. A method according to Claim 1, wherein said chelating agent is linked to said oligonucleotide through a linking chain of from 4 to 16 atoms in the chain.

6. A method according to Claim 1, wherein said cells are mammalian cells.

7. A method according to Claim 1, wherein said composition further comprises a moiety imparting amphiphilic properties to said composition, said moiety comprising at least 6 carbon atoms and being hydrophobic or amphiphilic.

8. A method of inhibiting the utilization of mRNA in a cellular process in vitro, said method comprising:
contacting cells in vitro with a composition capable of entering contacted cells, said composition comprising an oligonucleotide chelate conjugate capable of hybridiz-ing to an intracellular mRNA sequence, said oligonucleotide chelate conjugate comprising an oligonucleotide conjugated to a metal-ion free polyamino-polycarboxylic acid chelating agent capable of chelating a metal ion which is capable of initiating a reaction that results in the inactivation of said RNA sequence.

9. A method according to Claim 8, wherein said chelating agent is diethylene triamine pentaacetic acid.

10. A method according to Claim 9, wherein said chelating agent is joined to said oligonucleotide through a linking chain of from 4 to 16 atoms in the chain and through an amide functionality.

11. A method according to Claim 8, wherein said oligonucleotide is a polyphosphate.

12. A method according to Claim 8, wherein said oligonucleotide is a polyphosphonate.

13. A method according to Claim 8, wherein said oligonucleotide has from about 8 to 33 units.

14. A method according to Claim 8, wherein said composition further comprises a moiety imparting amphiphilic properties to said composition, said moiety comprising at least 6 carbon atoms and being hydrophobic or amphiphilic.

15. The use of a composition to inhibit the utilization of RNA in a cellular process, said composition being capable of entering cells contacted with the composition and said composition comprising an oligonucleotide chelate conjugate capable of hybridizing to a intracellular RNA sequence, said oligonucleotide chelate conjugate comprising an oligonucleotide conjugated to a metal-ion free chelating agent capable of chelating a metal ion which is capable of initiating a reaction that results in the inactivation of said RNA sequence.

16. The use according to Claim 15, wherein said oligonucleotide is at least about 50% G + C.

17. The use according to Claim 15, wherein said chelating agent is a polyamino-polycarboxylic acid chelating agent.

18. The use according to Claim 17, wherein said chelating agent is an alkylenepolyamine polycarboxymethylene chelating agent.

19. The use according to Claim 15, wherein said chelating agent is linked to said oligonucleotide through a linking chain of from 4 to 16 atoms in the chain.

20. The use according to Claim 15, wherein said cells are part of a mammalian host.

21. The use according to Claim 15, wherein said composition further comprises a moiety imparting amphiphilic properties to said composition, said moiety comprising at least 6 carbon atoms and being hydrophobic or amphiphilic.

22. The use of a composition to inhibit the utilization of mRNA in a cellular process, said composition being capable of entering contacted cells and said composition comprising an oligonucleotide chelate conjugate capable of hybridizing to an intracellular mRNA sequence, said oligonucleotide chelate conjugate comprising an oligonucleotide conjugated to a metal-ion free polyamino-polycarboxylic acid chelating agent capable of chelating a metal ion which is capable of initiating a reaction that results in the inactivation of said RNA sequence.

23. The use according to Claim 22, wherein said chelating agent is diethylene triamine pentaacetic acid.

24. The use according to Claim 23, wherein said chelating agent is joined to said oligonucleotide through a linking chain of from 4 to 16 atoms in the chain and through an amide functionality.

25. The use according to Claim 22, wherein said oligonucleotide is a polyphosphate.

26. The use according to Claim 22, wherein said oligonucleotide is a polyphosphonate.

27. The use according to Claim 22, wherein said oligonucleotide has from about 8 to 30 units.

28. The use according to Claim 22, wherein said composition further comprises a moiety imparting amphiphilic properties to said composition, said moiety comprising at least 6 carbon atoms and being hydrophobic or amphiphilic.