Patent Publication Number: US-5834607-A

Title: Amines and methods of making and using the same

Description:
This application is a continuation of application Ser. No. 07/943,516, filed Sep. 11, 1992, now abandoned, which is a continuation-in-part of application Ser. No. 07/558,663, files Jul. 27, 1990 (now U.S. Pat. No. 5,138,045) and of application Ser. No. 07/844,845, filed Mar. 3, 1992 (now U.S. Pat. No. 5,218,105). 
    
    
     FIELD OF THE INVENTION 
     This invention relates to novel amine-containing compounds useful for therapeutics and methods of making and using the same. 
     BACKGROUND OF THE INVENTION 
     It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. These interactions involved the binding of complementary &#34;antisense&#34; oligonucleotides or their analogs to the transcellular RNA in a sequence specific fashion such as by Watson-Crick base pairing interactions. 
     The pharmacological activity of antisense compounds, as well as other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor is the ability of antisense compounds to traverse the plasma membrane of specific cells involved in the disease process. 
     Cellular membranes consist of lipid protein bilayers that are freely permeable to small, nonionic, lipophilic compounds and inherently impermeable to most natural metabolites and therapeutic agents. Wilson, D. B. Ann. Rev. Biochem. 47:933-965 (1978). The biological and antiviral effects of natural and modified oligonucleotides in cultured mammalian cells have been well documented, so it appears that these agents can penetrate membranes to reach their intracellular targets. Uptake of antisense compounds into a variety of mammalian cells, including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1, and ATH8 cells has been studied using natural oligonucleotides and nuclease resistant analogs, such as alkyl triesters, Miller, P. S., Braiterman, L. T. and Ts&#39;O, P. O. P., Biochemistry 16:1988-1996 (1977); methylphosphonates, Marcus-Sekura, C. H., Woerner, A. M., Shinozuka, K. Zon, G., and Quinman, G. V. , Nuc. Acids Res. 15:5749-5763 (1987) and Miller, P. S., McParland, K. B., Hayerman, K. and Ts&#39;O, P. O. P., Biochemistry 20:1874-1880 (1981); and phosphorothioates, Ceruzzi, M. and Draper, K. Nucleosides &amp; Nucleotides 8:815-818 (1989); Miller, P. S., Braiterman, L. T. and Ts&#39;O, P. O. P. Biochemistry 16:1988-1996 (1977) and Loke, S. L., Stein, C., Zhang, X. H. Avigan, M., Cohen, J. and Neckers, L. M. Curr. Top. Microbiol. Immunol. 141:282-289 (1988). 
     Enhanced cellular uptake has previously been achieved by attachment of functional groups to the 3&#39; and 5&#39; end of oligonucleotides to enhance cellular uptake in specific cell types. Previous studies have shown that plasmid DNA complexed with an (asialo)glycoprotein-poly(L-lysine) conjugate, could be targeted to hepatocytes, which contain unique cell surface receptors for galactose-terminal (asialo)glycoproteins. Wu, G. Y. and Wu, C. H. Biochemistry 27:887-892 (1988). Other groups have synthesized oligodeoxyribonucleotides that have a 5&#39;-attached alkylating agent and a 3&#39; attached cholesterol moiety and determined that these modified oligonucleotides were taken up into cells more efficiently than control compounds without the steroid moiety. Zon, G. in Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression 234-247, ed. J. S. Cohen (CRC Press, Boca Raton Fla., 1989). Letsinger, et al. Proc. Natl. Acad. Sci. U.S.A. 86:653-656 (1989), have also synthesized cholesteryl-conjugated phosphorothioates whose anti-HIV activity is significantly greater than natural oligonucleotides with the same sequence. Additional modifications include conjugation of oligonucleotides to poly(L-lysine) alone. Stevenson, M. and Iversen, P. L. J. Gen. Virol 70:2673-2682 (1989) and Lemaitre, M., Baynard, B. and LeBleu, B. Proc. Natl. Acad. Sci. U.S.A. 84:648-652 (1987). This modification enhanced the antiviral activity of the compound studied presumably due to increased cellular uptake imparted by the polycationic poly(L-lysine). 
     The conjugation of polyamines to oligonucleotides have been found to enhance cellular uptake of oligonucleotides, increased lipophilicity, cause greater cellular retention and increased distribution of the compound. Vasseur, et al., Nucleosides and Nucleotides, 1991, 10, 107 prepared abasic sites at different sites of oligothymidylates by acid hydrolysis. Thereafter the abasic sites were functionalized with functionalities such as 3-amino carbazole, 9-amino elipticine and psoralen. Vasseur, et al. also refers to unpublished results in which the functionalities spermidine and proflavin were employed. Le Doan, et al., Nucleic Acids Research 1987, 15, 8643 teaches oligothymidylates covalently linked to porphyrins at their 3&#39; end via one of the linkers --O----CH 2  --CO--NH--(CH 2 ) 2  --NH or PO 4  --(CH 2 ) 6  --NH--. Le Doan, et al. also used the linker PO 4  --(CH 2 ) 6  --NH-- to link porphyrins to the 5&#39; end of oligothymidylates. Another group, Summerton, et al., U.S. Pat. No. 5,034,506 issued Jul. 23, 1991 teaches morpholino subunits, linked together by uncharged, achiral linkages such as amides. As described in PCT/US91/04086 filed Jun. 10, 1991, polyamines have also been linked at the 5&#39; end of an oligonucleotide at the 5&#39; site of the sugar moiety of the terminal nucleoside and at the 2-position carbon of the heterocyclic baseof 2&#39;-deoxyadenosine, 2&#39;-deoxyguanosines and other purines and purine analogs by known procedures as described in PCT/US/91/00243 filed Jan. 11, 1991. 
     Novel amines and methods of preparing the same are greatly needed in order to enhance cellular uptake of oligonucleotides, increase lipophilicity, cause greater cellular retention and increase distribution of the compound within the cell. The present invention fulfills this need. 
     OBJECTS OF THE INVENTION 
     It is one object of the present invention to provide novel amine-containing compounds useful in therapeutics. 
     It is a further object of the present invention to provide methods of producing said novel compounds. 
     It is another object of the present invention to provide methods of modulating the production of a protein by an organism. 
     It is still a further object of the present invention to provide methods of treating a mammal suffering from a disease characterized by the undesired production of a protein. 
     It is yet a further object of the present invention to provide methods of diagnosing the presence of an RNA in a biological sample. 
     These and other objects will become apparent from the following description and accompanying claims. 
     SUMMARY OF THE INVENTION 
     The present invention provides compounds which may have enhanced efficacy as an antisense-based therapy. Compounds of the present invention can have enhanced cellular uptake, increased lipophilicity, cause greater cellular retention and demonstrate increased distribution. Furthermore the present invention provides simple methods for synthesis of these novel compounds. 
     In accordance with some embodiments of the present invention, compounds having the structure: ##STR1## wherein R 1  and R 2  are independently H, a nucleotide, oligonucleotide, or polyamine and at least one of R 1  and R 2  is a purine containing oligonucleotide, R 3  is a linear or cyclic non-aromatic polyamine species, and X is H, O--R 11 , S--R 11 , F, Cl, Br, CN, CF 3 , OCF 3 , OCN, SOCH 3 , SO 2  CH 3 , ONO 2 , N 3 , HN 2 , heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R 11  is H, C 1  to C 10  straight or branched chain lower alkyl or substituted lower alkyl, C 2  to C 10  straight or branched chain lower alkenyl or substituted lower alkylenyl, C 3  to C 10  straight or branched chain lower alkynyl or substituted lower alkynyl, a  14  C containing lower alkyl, lower alkenyl or lower alkynyl, C 7  to C 14  substituted or unsubstituted alkylaryl or aralkyl, a  14  C containing C 7  to C 14  alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, are provided. 
     In accordance with still other embodiments of the present invention, compounds having the structure: ##STR2## wherein R 4  is an oligonucleotide and M is a pendent group having a polyamine species attached thereto are provided. 
     Methods of preparing such compounds utilizing enzymatic reagents are also provided in some aspects of the invention. Thus compounds of Formula I may be prepared by methods comprising the steps of providing a synthon having the structure: ##STR3## wherein R 1  and R 2  are independently H, a nucleotide, oligonucleotide or polyamine, and at least one of R 1  and R 2  is a purine containing oligonucleotide, and X is H, O--R 11 , S--R 11 , F, Cl, Br, CN, CF 3 , OCF 3 , OCN, SOCH 3 , SO 2  CH 3 , ONO 2 , N 3 , HN 2 , heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R 11  is H, C 1  to C 10  straight or branched chain lower alkyl or substituted lower alkyl, C 2  to C 10  straight or branched chain lower alkenyl or substituted lower alkylenyl, C 3  to C 10  straight or branched chain lower alkynyl or substituted lower alkynyl, a  14  C containing lower alkyl, lower alkenyl or lower alkynyl, C 7  to C 14  substituted or unsubstituted alkylaryl or aralkyl, a  14  C containing C 7  to C 14  alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide. Thereafter the synthon is reacted with R 3 , wherein R 3  is a linear or cyclic non-aromatic polyamine species, under reducing conditions to yield the final product. 
     Compounds of Formula II may also be prepared enzymatically by providing a starting material having the structure: ##STR4## wherein R 4  is an oligonucleotide, R 12  is an oligonucleotide and B is urea or a heterocyclic base having a corresponding glycosylase and reacting the starting material with an endonuclease to generate a conjugated α,β-unsaturated system in the sugar residue of the 3&#39; terminal nucleotide. Thereafter the compound having a conjugated α,β-unsaturated system is reacted with a pendent group containing a nucleophile functionality thereon. Following addition of the pendent group the double bond of the α,β system is reduced with a reducing agent. A polyamine species may then be attached to the pendent group via an alkylation reaction. Alternatively, a polyamine species may be attached to a pendent group which is a bifunctional linker. 
     In accordance with still other embodiments of the present invention compounds having the structure: ##STR5## wherein R 4  is an oligonucleotide, R 5  is a linear or cyclic non-aromatic polyamine species containing at least one non-amide nitrogen atom, and R 6  is H, a purine heterocycle or a pyrimidine heterocycle, are provided. Methods of preparing compounds of Formula III are also provided in some aspects of the present invention comprising the steps of reacting an oligonucleotide having a 3&#39; ribofuranosyl sugar with an oxidizing agent to produce an activated dialdehyde-terminated oligonucleotide and reacting said activated oligonucleotide with a linear or cyclic non-aromatic polyamine species under reducing conditions to yield said compound. 
     In accordance with other aspects of the invention compounds having the structure: ##STR6## wherein B is a purine or pyrimidine heterocyclic base, R 8  and R 9  are independently H, PO 2 , a nucleotide, oligonucleotide or polyamine species and at least one of R 8  and R 9  is a purine containing oligonucleotide, and at least one of R 8 , R 9  and A is a species comprising the formula L 1  -L 2  -polyamine wherein L 1  is an amino linker and L 2  is a heterobifunctional linker; and wherein if R 8  is not a purine containing oligonucleotide or polyamine species, then R 8  is a nucleotide or PO 2  ; if R 9  is not a purine containing oligonucleotide or polyamine species, then R 9  is H or a nucleotide; and if A is not a polyamine species then A is H or OH are provided. 
     Therapeutic and diagnostic methods are also encompassed by the present invention. Methods of modulating the production of protein by an organism comprising contacting an organism with a compound having the structure of Formula I, Formula II, Formula III or Formula IV are encompassed by some embodiments of the present invention. In other aspects of the invention, methods of treating an animal having a disease characterized by undesired production of protein comprising contacting an animal with a compound having the structure of Formula I, Formula II, Formula III, or Formula IV in a pharmaceutically acceptable carrier are provided. Still other methods of the present invention provide methods for detecting the presence or absence of an RNA in a biological sample suspected of containing said RNA are provided comprising contacting a sample with a compound having the structure of Formula I, Formula II, Formula III or Formula IV wherein the compound is specifically hybridizable with the RNA and detecting the presence or absence of hybridization of the compound to the sample wherein hybridization is indicative of the presence of RNA in the sample. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 is a schematic representation of one preferred syntheses of compounds of Formula I. 
     FIG. 2 is a schematic representation of one preferred syntheses of compounds of Formula II. 
     FIG. 3 is a schematic representation of one preferred syntheses of compounds of Formula III. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     The present invention provides novel amine compounds useful for antisense therapy. In one embodiment of the present invention compounds having the structure: ##STR7## wherein R 1  and R 2  are independently H, a nucleotide, oligonucleotide, or polyamine and at least one of R 1  and R 2  is a purine containing oligonucleotide, R 3  is a linear or cyclic non-aromatic polyamine species, and X is H, O--R 11 , S--R 11 , F, Cl, Br, CN, CF 3 , OCF 3 , OCN, SOCH 3 , SO 2  CH 3 , ONO 2 , N 3 , HN 2 , heterocylcoalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R 11  is H, C 1  to C 10  straight or branched chain lower alkyl or substituted lower alkyl, C 2  to C 10  straight or branched chain lower alkenyl or substituted lower alkylenyl, C 3  to C 10  straight or branched chain lower alkynyl or substituted lower alkynyl, a  14  C containing lower alkyl, lower alkenyl or lower alkynyl, C 7  to C 14  substituted or unsubstituted alkylaryl or aralkyl, a  14  C containing C 7  to C 14  alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, are provided. In some embodiments of the present invention both R 1  and R 2  are oligonucleotides, at least one of which includes at least one purine nucleotide. 
     In the context of this invention, the term &#34;oligonucleotide&#34; refers to a polynucleotide formed from naturally occurring bases, such as purine and pyrimidine heterocycles, and furanyl groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or synthetic species formed from naturally occurring subunits or their close homologs. The term &#34;oligonucleotide&#34; may also refer to moieties which have portions similar to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur-containing species which are known for use in the art. In accordance with some preferred embodiments, at least some of the phosphodiester bonds of the oligonucleotide have been substituted with a structure which functions to enhance the stability of the oligonucleotide or the ability of the oligonucleotide to penetrate into the region of cells where the viral RNA is located. It is preferred that such substitutions comprise phosphorothioate bonds, phosphotriesters, methyl phosphonate bonds, short chain alkyl or cycloalkyl structures or short chain heteroatomic or heterocyclic structures. Most preferred are CH 2  --NH--O--CH 2 , CH 2  --N(CH 3 )--O--CH 2 , CH 2  --O--N(CH 3 )--CH 2 , CH 2  --N(CH 3 )--N(CH 3 )--CH 2  and O--N(CH 3 )--CH 2  --CH 2  structures where phosphodiester is O--P--O--CH 2 ). Also preferred are morpholino structures. Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506 issued Jul. 23, 1991. In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replace with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. P. E. Nielsen, et al., Science 1991 254 1497. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with other structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Still other linkages include the those disclosed in U.S. patent applications Ser. No. 566,836, filed Aug. 13, 1990, entitled Novel Nucleoside Analogs; Ser. No. 703,619, filed May 21, 1991, entitled Backbone Modified Oligonucleotide Analogs; Ser. No. 903,160, filed Jun. 24, 1992, entitled Heteroatomic Oligonucleoside Linkages; Ser. No. PCT/US92/04294, filed May 21, 1992, entitled Backbone Modified Oligonucleotides; and Ser. No. PCT/US92/04305, all assigned to the assignee of this invention. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention. 
     Oligonucleotides may also include species which include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. For example, deaza or aza purines and pyrimidines may be used in place of naturally purine or pyrimidine bases and pyrimidine bases having substituent groups at the 5- or 6-positions; purine bases having altered or replacement substituent groups at the 2-, 6- or 8-positions are also provided in some aspects of the present invention. Similarly, modifications on the furanosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2&#39;--O-alkyl- and 2&#39;-halogen-substituted nucleotides. Some specific examples of modifications at the 2&#39; position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 , F, OCN, O(CH 2 ) n  NH 2 , O(CH 2 ) n  CH 3  where n is from 1 to about 10; C 1  to C 10  lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl, Br, CN, CF 3 , OCF 3 , O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 , SO 2  CH 3  ; ONO 2  ; NO 2  ; N 3  ; NH 2  ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Sugar mimetics such as cyclobutyls may also be used in place of the pentofuranosyl group. Exemplary modifications are disclosed in U.S. patent applications: Ser. No. 463,358, filed Jan. 11, 1990, entitled Compositions And Methods For Detecting And Modulating RNA Activity; Ser. No. 566,977, filed Aug. 13, 1990, entitled Sugar Modified Oligonucleotides That Detect And Modulate Gene Expression; Ser. No. 558,663, filed Jul. 27, 1990, entitled Novel Polyamine Conjugated Oligonucleotides; Ser. No. 558,806, filed Jul. 27, 1991, entitled Nuclease Resistant Pyrimidine Modified Oligonucleotides That Detect And Modulate Gene Expression; and Ser. No. PCT/US91/00243, filed Jan. 11, 1991, entitled Compositions and Methods For Detecting And Modulating RNA Activity; Ser. No. 777,670, filed Oct. 15, 1991, entitled Oligonucleotides Having Chiral Phosphorus Linkages; Ser. No. 814,961, filed Dec. 24, 1991, entitled Gapped 2&#39; Modified Phosphorothioate Oligonucleotides; Ser. No. 808,201, filed Dec. 13, 1991, entitled Cyclobutyl Oligonucleotide Analogs; and Ser. No. 782,374, filed 782,374, entitled Derivatized Oligonucleotides Having Improved Uptake &amp; Other Properties, all assigned to the assignee of this invention. The disclosures of all of the above noted patent applications are incorporated herein by reference. Oligonucleotides may also comprise other modifications consistent with the spirit of this invention. Such oligonucleotides are best described as being functionally interchangeable with yet structurally distinct from natural oligonucleotides. All such oligonucleotides are comprehended by this invention so long as they effectively function as subunits in the oligonucleotide. Thus, purine containing oligonucleotide are oligonucleotides comprising at least one purine base or analog thereof. In other embodiments of the present invention compounds of the present invention may be &#34;subunits&#34; of a species comprising two or more compounds of the present invention which together form a single oligonucleotide. 
     Oligonucleotides of the present invention may be naturally occurring or synthetically produced and may range in length from about 8 to about 50 nucleotides. In more preferred embodiments of the present invention said oligonucleotides may be from 8 to 40 nucleotides in length. Most preferably, oligonucleotides of the present invention may be from 12 to about 20 nucleotides in length. 
     The phrase polyamine species as used throughout the specification refers to species that have a plurality of nitrogen atoms thereon. Polyamines include primary amines, hydrazines, semicarbazines, thiosemicarbazines and similar nitrogenous species. Such species can be symmetrical species such as polyamine containing polymers or they can be unsymmetrical wherein the amine functionalities of the polyamine are separated in space by different moieties. In addition to carbon atoms other atomic species such as nitrogen and sulfur may also be incorporated into the polyamine species. In some preferred embodiments of the invention, at least one nitrogen atom of the polyamine has a free electron pair. 
     Preferred as polyamine species are species that range in length from about 3 to about 20 units. More preferably species having at least one nitrogen atom have the general formula H 2  N (CH 2 ) n  NH! m  -- wherein n is an integer between 2 and 8 and m is an integer between 1 and 10. These species can be linear or cyclic. Cyclic amines would include crown amines and mixed crown amines/crown ethers. 
     Other suitable nitrogen-containing compound suitable for the formation of polyamine species include C 1  -C 20  straight chain alkylamine, C 1  -C 20  straight chain substituted alkylamine, C 2  -C 50  branched chain alkylamine, C 2  -C 50  branched chain substituted alkylamine, C 3  -C 50  cyclic alkylamine, C 3  -C 50  cyclic substituted alkylamine, C 2  -C 20  straight chain alkenylamine, C 2  -C 20  straight chain substituted alkenylamine, C 3  -C 50  branched chain alkenylamine, C 3  -C 50  branched chain substituted alkenylamine, C 3  -C 50  cyclic alkenylamine, C 3  -C 50  cyclic substituted alkenylamine, C 2  -C 20  straight chain alkynylamine, C 2  -C 20  straight chain substituted alkynylamine, C 3  -C 50  branched chain alkynylamine, C 3  -C 50  branched chain substituted alkynylamine, C 3  -C 50  cyclic alkynylamine, C 3  -C 50  cyclic substituted alkynylamine, C 1  -C 20  straight chain alkylhydrazine, C 1  -C 50  straight chain substituted alkylhydrazine, C 2  -C 50  branched chain alkylhydrazine, C 2  -C 50  branched chain substituted alkylhydrazine,  3  -C 50  cyclic hydrazoalkane, C 3  -C 50  cyclic substituted hydrazoalkane, C 2  -C 20  straight chain alkenylhydrazine, C 2  -C 20  straight chain substituted alkenylhydrazine, C 3  -C 50  branched chain alkenylhydrazine, C 3  -C 50  branched chain substituted alkenylhydrazine, C 3  -C 50  cyclic hydrazoalkene, C 3  -C 50  cyclic substituted hydrazoalkene,C 2  -C 20  straight chain alkynylhydrazine, C 2  -C 20  straight chain substituted alkynylhydrazine, C 3  -C 50  branched chain alkynylhydrazine, C 3  -C 50  branched chain substituted alkynylhydrazine, C 3  -C 50  cyclic hydrazoalkyne, C 3  -C 50  cyclic substituted hydrazoalkyne, C 1  -C 20  straight chain alkylhydroxyamine, C 1  -C 20  straight chain substituted alkylhydroxyamine, C 2  -C 50  branched chain alkylhydroxyamine, C 2  -C 50  branched chain substituted alkylhydroxyamine, C 3  -C 50  cyclic oxyalkylamine, C 3  -C 50  cyclic substituted oxyalkylamine, C 2  -C 20  straight chain alkenylhydroxyamine, C 2  -C 20  straight chain substituted alkenylhydroxyamine, C 3  -C 50  branched chain alkenylhydroxyamine, C 3  -C 50  branched chain substituted alkenylhydroxyamine, C 3  -C 50  cyclic oxyalkenylamine, C 3  -C 50  cyclic substituted oxyalkenylamine, C 2  -C 20  straight chain alkynylhydroxyamine, C 2  -C 20  straight chain substituted alkynylhydroxyamine, C 3  -C 50  branched chain alkynylhydroxyamine, C 3  -C 50  branched chain substituted alkynylhydroxyamine, C 3  -C 50  cyclic oxyalkynylamine, C 3  -C 50  cyclic substituted oxyalkynylamine, C 1  -C 20  straight chain alkylsemicarbazide, C 1  -C 20  straight chain substituted alkylsemicarbazide, C 2  -C 50  branched chain alkylsemicarbazide, C 2  -C 50  branched chain substituted alkylsemicarbazide, C 3  -C 50  cyclic alkylsemicarbazide, C 3  -C 50  cyclic substituted alkylsemicarbazide, C 2  -C 20  straight chain alkenylsemicarbazide, C 2  -C 20  straight chain substituted alkenylsemicarbazide, C 3  -C 50  branched chain alkenylsemicarbazide, C 3  -C 50  branched chain substituted alkenylsemicarbazide, C 3  -C 50  cyclic alkenylsemicarbazide, C 3  -C 50  cyclic substituted alkenylsemicarbazide, C 2  -C 20  straight chain alkynylsemicarbazide, C 2  -C 20  straight chain substituted alkynylsemicarbazide, C 3  -C 50  branched chain alkynylsemicarbazide, C 3  -C 50  branched chain substituted alkynylsemicarbazide, C 3  -C 50  cyclicalkynylsemicarbazide, C 3  -C 50  cyclic substituted alkynylsemicarbazide, C 1  -C 20  straight chain alkylthiosemicarbazide, C 1  -C 20  straight chain substituted alkylthiosemicarbazide, C 2  -C 50  branched chain alkylthiosemicarbazide, C 2  -C 50  branched chain substituted alkylthiosemicarbazide, C 3  -C 50  cyclic alkylthiosemicarbazide, C 3  -C 50  cyclic substituted alkylthiosemicarbazide, C 2  -C 20  straight chain alkenylthiosemicarbazide, C 2  -C 20  straight chain substituted alkenylthiosemicarbazide, C 3  -C 50  branched chain alkenylthiosemicarbazide, C 3  -C 50  branched chain substituted alkenylthiosemicarbazide, C 3  -C 50  cyclic alkenylthiosemicarbazide, C 3  -C 50  cyclic substituted alkenylthiosemicarbazide, C 2  -C 20  straight chain alkynylthiosemicarbazide, C 2  -C 20  straight chain substituted alkynylthiosemicarbazide, C 3  -C 50  branched chain alkynylthiosemicarbazide, C 3  -C 50  branched chain substituted alkynylthiosemicarbazide, C 3  -C 50  cyclic alkynylthiosemicarbazide, C 3  -C 50  cyclic substituted alkynylthiosemicarbazide, C 1  -C 20  straight chain alkylhydrazone, C 1  -C 20  straight chain substituted alkylhydrazone, C 2  -C 50  branched chain alkylhydrazone, C 2  -C 50  branched chain substituted alkylhydrazone, C 3  -C 50  cyclic hydrazoalkane, C 3  -C 50  cyclic substituted hydrazoalkane, C 2  -C 20  straight chain alkenylhydrazone, C 2  -C 20  straight chain substituted alkenylhydrazone, C 3  -C 50  branched chain alkenylhydrazone, C 3  -C 50  branched chain substituted alkenylhydrazone, C 3  -C 50  cyclic hydrazoalkene, C 3  -C 50  cyclic substituted hydrazoalkene, C 2  -C 20  straight chain alkynylhydrazone, C 2  -C 20  straight chain substituted alkynylhydrazone, C 3  -C 50  branched chain alkynylhydrazone, C 3  -C 50  branched chain substituted alkynylhydrazone, C 3  -C 50  cyclic hydrazoalkyne, C 3  -C 50  cyclic substituted hydrazoalkyne, C 1  -C 20  straight chain alkylhydrazide, C 1  -C 20  straight chain substituted alkylhydrazide, C 3  -C 50  branched chain alkylhydrazide, C 3  -C 50  branched chain substituted alkylhydrazide, C 3  -C 50  cyclic alkylhydrazide, C 3  -C 50  cyclic substituted alkylhydrazide, C 2  -C 20  straight chain alkenylhydrazide, C 2  -C 20  straight chain substituted alkenylhydrazide, C 3  -C 50  branched chain alkenylhydrazide, C 3  -C 50  branched chain substituted alkenylhydrazide, C 3  -C 50  cyclic alkenylhydrazide, C 3  -C 50  cyclic substituted alkenylhydrazide, C 2  -C 20  straight chain alkynylhydrazide, C 2  -C 20  straight chain substituted alkynylhydrazide, C 3  -C 50  branched chain alkynylhydrazide, C 3  -C 50  branched chain substituted alkynylhydrazide, C 3  -C 50  cyclic alkynylhydrazide and C 3  -C 50  cyclic substituted alkynylhydrazide. 
     In accordance with preferred embodiments of the present invention polyamine species are linear or cyclic non-aromatic polyamine species. In still more preferred embodiments of the present invention polyamine species are linear or cyclic non-aromatic comprising non-amide nitrogen atoms. By non-amide is meant a nitrogen which is not adjacent to a carbonyl group (i.e. C═O or C═S). 
     In still other embodiments of the present invention compounds having the structure: ##STR8## wherein R 4  is an oligonucleotide and M is a pendent group having a polyamine species attached thereto are provided. The pendent group may be any group to which a polyamine may be attached. In preferred embodiments the pendent group is a R 10  S or R 10  NH, wherein R 10  is any of a broad range of reactive groups effective for subsequent attachment of polyamine species to the pendent group. Suitable for R 10  are substituted and un-substituted, straight chain or branched chained C 1  -C 20  alkyl groups or substituted or un-substituted C 7  -C 14  aryl groups having the nucleophile in one position thereon and a further functional group in a further position thereon. The pendent group may thus, subsequently functionalized with a bifunctional linker group amendable for attachment of a polyamine species to the pendent group. Alternatively the polyamine species may be directly attached to a pendent group such as by alkylation. 
     Further in accordance with the present invention are provided compounds having the structure: ##STR9## wherein R 4  is an oligonucleotide, R 5  is a linear or cyclic non-aromatic polyamine species containing non-amide nitrogen atoms, and R 6  is H, a purine heterocycle or a pyrimidine heterocycle. 
     The present invention also provides novel polyamine containing compounds having the structure: ##STR10## wherein B is a purine or pyrimidine heterocycle, R 8  and R 9  are independently H, PO 2   - , a nucleotide, oligonucleotide or polyamine species and at least one of R 8  and R 9  is a purine containing oligonucleotide, and at least one of R 8 , R 9  and A is a species comprising the formula L 1  -L 2  -polyamine wherein L 1  is an amino linker and L 2  is a heterobifunctional linker; and wherein if R 8  is not a purine containing oligonucleotide or polyamine species, then R 8  is a nucleotide or PO 2   -  ; if R 9  is not a purine containing oligonucleotide or polyamine species, then R 9  is H or a nucleotide; and if A is not a polyamine species then A is H or OH. 
     Thus R 8  and R 9  may be oligonucleotides and A may be a species comprising the formula L 1  -L 2  -polyamine, or alternatively, R 8  may be an oligonucleotide and one or both of R 9  and A may be a species comprising the formula L 1  -L 2  -polyamine; or R 9  may be an oligonucleotide and one or both of R 8  and A may be a species comprising the formula L 1  -L 2  -polyamine. Furthermore, when R 8  is not a purine containing oligonucleotide or polyamine species, then R 8  is a nucleotide or PO 2   - . If R 9  is not a purine containing oligonucleotide or polyamine species, then R 9  is H or a nucleotide, and if A is not a polyamine species then A is H or OH. 
     In preferred embodiments of the present invention commercially available amino linkers may be used. For example, the 3&#39; amino modifiers having the trade names C3 CPG and C7 CPG available through Glen Research may be employed. 5&#39; amino modifiers may also be used such as C3 and C7 5&#39; branched modifiers available through Glen Research. Similarly, 2&#39; amino modifiers are also envisioned for use in some aspects of the present invention. The amino linkers are designed to functionalize a target oligonucleotide by the introduction of a primary amine at a designated site, be it 2&#39;, 3&#39; or 5&#39;. As will be apparent to one skilled in the art, any linker which meets this end is encompassed by the present invention. 
     Likewise, bifunctional linkers effective for purposes of the present invention are available commercially. For example, bis-(maleimido)-methyl ether (BMME), disuccinimidyl suberate (DSS), 3-maleimidobenzoyl-N-hydroxy-succinimide (MBS), maleimidohexanoyl-N-hydroxyl-succinimide (MHS) and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) may be useful in some embodiments of the present invention. Other useful bifunctional linkers will be apparent to one skilled in the art as for instance from Pierce, Rockford, Ill. 
     Compounds of the present invention may be prepared by providing an oligonucleotide comprising one or more abasic sites. In the context of the present invention &#34;abasic site&#34; refers to a nucleotide unit in which the purine or pyrimidine group has been removed or replaced by a non-naturally occurring group such as a hydroxyl group. One or more abasic sites may be incorporated into one or more nucleotide bases of an oligonucleotide to form a synthon having the structure: ##STR11## wherein R 1  and R 2  are independently H, a nucleotide, oligonucleotide or polyamine species and at least one of R 1  and R 2  is a purine containing oligonucleotide, and X is H, O--R 11 , S--R 11 , F, Cl, Br, CN, CF 3 , OCF 3 , OCN, SOCH 3 , SO 2  CH 3 ,ONO 2 , N 3 , HN 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter molecule, an RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide wherein R 11  is H, C 1  to C 10  straight or branched chain lower alkyl or substituted lower alkyl, C 2  to C 10  straight or branched chain lower alkenyl or substituted lower alkylenyl, C 3  to C 10  straight or branched chain lower alkynyl or substituted lower alkynyl, a  14  C containing lower alkyl, lower alkenyl or lower alkynyl, C 7  to C 14  substituted or unsubstituted alkylaryl or aralkyl, a  14  C containing C 7  to C 14  alkaryl or aralkyl, alicyclic, heterocyclic, a reporter molecule, a RNA cleaving group, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide. 
     An enzymatic process may be used to produce such a synthon having abasic sites by reaction of a DNA glycosylase with an oligonucleotide starting material. For example, uracil DNA glycosylase act on uracil bases within an oligonucleotide to create abasic sites. Of course it should be recognized that enzymatic methods using DNA glycosylase may be less effective for oligonucleotides more closely resembling RNA such as oligonucleotides having 2&#39; modifications. 
     Enzymes, as employed in the present invention, may be derived from naturally occurring sources or may be prepared by recombinant techniques. Many useful enzymes are available commercially. 
     Such synthons may alternatively be prepared by incorporation of abasic sites into an oligonucleotide via abasic sugar precursors. For example, 5-O-(4,4&#39;-dimethoxytrityl)-1,2-dideoxy-1-(o-nitrobenzoyl)-D-ribofuranose-3-O-(2-Cyanoethyl-N,N&#39;-diisopropyl) phosphoramidite may be prepared by modification of the procedures of Lyer, et al., Nucleic Acids Research 18: 2855 (1990) and Didier, et al., Tetrahedron Letters 32: 207 (1991). 
     Phosphoramidites having a 2&#39; substitutions and abasic sites may also be prepared. For example, a synthon may have 2&#39;-O-methyl or 2&#39;-fluoro substitutions. Such phosphoramidite may be incorporated into an oligonucleotide by standard procedures. An o-nitrobenzyldeoxyfuranose containing oligonucleotide can be synthesized in accordance with these procedures. Post synthesis photolysis utilizing a high intensity Hg lamp generates the corresponding abasic site-containing polymer. In addition, other methods of introducing abasic sites at the 3&#39;, 5&#39; and internal positions of an oligonucleotide to form a synthon are known to those skilled in the art. Thereafter the synthon may be reacted with a polyamine species under reducing conditions. As illustrated in FIG. 1, Step A, a compound may be prepared wherein B is uridine and an enzymatic process may be used to produce a synthon having abasic sites at one or more uridine sites by digestion of the compound with an enzyme such as uracil-DNA glycosylase. Other glycosylases will be effective for different targets. As described above, a glycosylase may be determined by the combined sequence of R 1 , R 2  and B. Some useful glycosylases and their respective targets are described for example, by Friedberg, DNA Repair (W. H. Freeman and Company, NY, 1985) p. 153, incorporated by reference herein in its entirety. These enzymes are commercially available or may be prepared from known procedures in the art. 
     In other embodiments of the present invention as exemplified in FIG. 2 compound of Formula II may be prepared by providing starting material having the structure: ##STR12## wherein R 4  is an oligonucleotide, R 12  is an oligonucleotide and B is urea or a heterocyclic base having a corresponding glycosylase and reacting the compound with an endonuclease to produce the compound 2 as described by Manoharan, et al., JACS, 1988, 110, 2690. Thereafter, the compound 2 is contacted with a pendent group such as R 10  S, and reduced with the reducing agent NaCNBH 4  to stabilize the product 5. A polyamine species may then be added such as by alkylation to provide the final product 7. A polyamine species may alternatively be added directly to a bifunctional pendent group. Some endonucleases which will be useful in embodiments of the present invention are described, for example, in Doetsch and Cunningham, Mutation Research, 1990, 236, 173, incorporated by reference herein in its entirety. The endonuclease chosen will depend upon the identity of B and the sequence of R 4  and/or R 12 . Thus, if B is a pyrimidine heterocycle, and the sequence of R 12  begins with a pyrimidine, then an endonuclease such as T4 or M. luteus UV endonuclease may be chosen. Following digestion by T4 or M. luteus UV endonuclease, B and R 12  are removed, resulting in a 3&#39; terminal α,β unsaturated aldehydic species. In some instances, it may be desireable to engineer the sequence of the species so as to provide a endonuclease digestion site at a desired location. 
     Thus, in one preferred embodiment of the present invention R 4  may be TGGGAGCCATAGCGAGGC (SEQ ID NO: 1), B may be the pyrimidine thymine and R 12  may be a thymidine dinucleotide. The net result of digestion of this species with T4 UV endonuclease will be TGGGAGCCATAGCGAGGCN (SEQ ID NO: 2) wherein N represents the aldehydic species. 
     Treatment of the digested compound with pendent group comprising a linker bearing a nucleophile results in the addition of the pendent group at the 3&#39; terminus of the compound to join the linker to the digested compound. Suitable nucleophilic species include thiols and amines moieties as described above. In preferred embodiments of the present invention the pendent group is R 10  S -   or R 10  NH. A polyamine species such as NH 2  (CH 2 ) n  NH 2  wherein n is an integer from 1 to about 10 could be used as the attacking nucleophile by suitably blocking one end thereof and utilizing the other end as the attacking nucleophilic species. R 10  can be further selected to provide a linkage or bridge between the nucleophile and a polyamine. Suitable for R 10  are substituted and un-substituted, straight chain or branched chained C 1  -C 20  alkyl groups or substituted or un-substituted C 7  -C 14  aryl groups having the nucleophile in one position thereon and a further functional group in a further position thereon. After attachment of the pendent group via nucleophilic attack on compound 2, for attachment of the polyamine species the further functional group is then derivitized either via a bifunctional linking group, an alkylation type reaction or other derivation reaction known to those skilled in the art. 
     Upon addition of the pendent group to the digested compound, the double bond remaining on the digested compound is reduced to stabilize the product. Reducing agents effective to stabilize the end product of such a reaction are well known in the art. Some suitable reducing agents include sodium cyanoborohydride, lithium cyanoborohydride and sodium borohydride. 
     Thereafter a polyamine may be added via an alkylation reaction or directly to a pendent group which is a bifunctional linker. The compound may further be derivatized by attaching one or more reactive groups to at least one of the nitrogen atoms of the polyamine species. Reactive groups include, but are not limited to reporter groups, alkylating agents, intercalating agents, RNA cleaving moieties, cell receptor binding molecules, steroids, peptides, crown amines, porphyrins and cross-linking agents. 
     In accordance with other methods of the present invention compounds of Formula III may be prepared by reacting an oligonucleotide having a 3&#39; ribofuranosyl sugar with an oxidizing agent to produce an dialdehyde-terminated activated oligonucleotide. Suitable oxidants include periodate solution, lead tetraacetate, activated MnO 2 , thallium (III) salts, pyridinium chlorochromate and O 2  catalyzed by Co (III) salts. 
     Thereafter the dialdehyde-terminated activated oligonucleotide is reacted with a polyamine species under reducing conditions. Reducing agents are known to those skilled in the art. Preferably, the activated oligonucleotide and species containing at least one nitrogen atom will be reacted in the presence of a solution of sodium cyanoborohydride, lithium cyanoborohydride or sodium borohydride. 
     In preferred embodiments of the present invention compounds may be produced as illustrated by FIG. 3, by preparation of an oligonucleotide having a 3&#39; ribofuranosyl end followed by attack of the 3&#39; ribofuranosyl ring by an oxidant such as m-periodate solution in 0.1M NaOac buffer pH5, as described by Bayard, Bisbal and Lebleu in Biochemistry 25: 3730 (1986) to produce a dialdehyde-terminated activated oligonucleotide (FIG. 3, Step A). The activated oligonucleotide and a species containing four nitrogen atoms, spermine, can be reacted in the presence of the reducing agent, sodium cyanoborohydride (FIG. 3, Step B). 
     Compounds of the present invention are preferably specifically hydridizable with a target region. By &#34;specifically hybridizable&#34; herein is meant capable of forming a stable duplex with a target DNA or RNA. It is believed that oligonucleotides which form Watson-Crick base pairs, i.e. are complementary with target DNA or RNA and which specifically hybridize with target DNA or RNA inhibit the flow of genetic information from DNA to protein. In some embodiments of the present invention the oligonucleotide portions of compounds of the present invention are at least 70% complementary to a target sequence. In preferred embodiments of the present invention the oligonucleotide portions of compounds of the present invention are at least 80% complementary to a target sequence. 100% complementarity of the oligonucleotide portions of compounds of the present invention to a target sequence is most preferred. In preferred embodiments of the present invention, the oligonucleotide portions may be specifically hybridizable with DNA or RNA from papilloma virus, herpes viruses, human immunodeficiency virus, Candida, cytomegaloviruses, and influenza viruses. In addition, the oligonucleotide portions may also be specifically hybridizable with endogenous DNA or RNA of a cell. By oligonucleotide portions is meant R 1  and/or R 2  of Formula I, R 4  of Formula II, R 4  and/or R 6  of Formula III, or R 8  and/or R 9  of Formula IV. 
     For therapeutics, an animal suspected of having a disease characterized by excessive or abnormal production of a protein is treated by administering a compound having the structure set forth in Formula I, Formula II, Formula III, or Formula IV in a pharmaceutically acceptable carrier. Most preferable, the compound is hybridizable with an RNA coding for the protein. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. Such treatment is generally continued until either a cure is effected or a diminution in the diseased state is achieved. Long term treatment is likely for some diseases. 
     The compounds of the present invention will also be useful as a research reagent useful for the modulation of the production of a protein by an organism. Modulation may be accomplished by contacting the organism with compounds of the present invention having structures as set forth in Formula I, Formula II, Formula III, or Formula IV. Preferably the compounds are hybridizable with RNA coding for the protein. 
     Diagnostic applications include the detection of the presence or absence of an RNA in a sample suspected of containing RNA comprising contacting the sample with a compound having structures as set forth in Formula I, Formula II, Formula III or Formula IV wherein the compound is specifically hybridizable with the RNA and detecting the presence or absence of hybridization of the compound to the sample wherein hybridization is indicative of the present of the RNA in the sample. 
     It is also envisioned by the present invention to provide compounds in which at least one of the nitrogen atoms of the polyamine are derivatized with one or more of the group consisting of functionalities such as reporter groups, alkylating agents, intercalating agents, cell receptor binding molecules, steroids, crown amines, porphyrins and cross-linking agents. Therapeutic, diagnostic and research reagent applications are equally, or even more effective when the polyamine species further comprises such groups. Such compounds allow greater numbers of functionalities to be delivered to a target. For example, reporter groups such as biotin, fluorescent molecules and various fluorophores may be attached to compounds of the present invention to effect diagnostic ends, resulting in signal amplification as compared to conventional oligonucleotide-reporter group combinations. In a preferred embodiment of the present invention, biotin may be used to functionalize compounds of the present invention by reacting a compound with D-biotin-N-hydroxysuccinimide ester. In a further preferred embodiment, the polyamine species may be further functionalized by reacting the compound containing the polyamine species with an activated ester having the structure: ##STR13## to form a compound with repeating imidazole catalytic cleaver units useful as an antisense therapeutic agents. Heterobifunctional linkers also can be utilized for attachment of intercalators, RNA cleaving agents including imidazoles, cell receptor binding molecules, steroids, alkylating agents, crown amines, porphyrins and cross-linkers to the polyamine species. 
     The following examples are illustrative but are not meant to be limiting of the present invention. 
     EXAMPLES 
     Example 1 
     Preparation of an Abasic Site Containing Oligonucleotide via Enzymatic Reaction 
     A. Synthesis of an Oligonucleotide Containing a Single Uridine Site 
     An oligonucleotide having the sequence CGC AGU CAG CC (SEQ ID NO:3) wherein U represents a 2&#39; deoxyuridine nucleotide, was prepared by standard solid phase synthesis. The deoxyuridine nucleotide in the middle of the sequence was added during synthesis utilizing deoxyuridine phosphoramidite (Glen Research, Sterling, Va.). The oligonucleotide was prepared utilizing standard synthesis cycles. It was deprotected by normal deprotection at 55° C. utilizing ammonium hydroxide, 30%, for 16 hours. The solvent was evaporated and the residue was purified by HPLC and detritylated. Final purification was effected on Sephadex G-25. 
     B. Preparation of Enzyme Stock Solution 
     Uracil-DNA glycosylase was isolated from E. Coli M5219 cells transformed with the expression plasmid pBD396 containing the ung gene. The enzyme was purified by electrophoretic homogeneity as described by Lindahl et al., J. Biol. Chem. 252: 3286 (1977) and stored in 30 mM HEPES-NaOH, pH 7.4, containing 5% glycerol, 2 mM DTT and 1 mM EDTA. 
     C. Preparation of Oligonucleotide Containing Single Abasic Site 
     An abasic oligonucleotide of the sequence CGC AGN CAG CC (SEQ ID NO:4) wherein N represents an abasic site, was prepared by treating 237 O.D. units of an oligonucleotide having SEQ ID NO:1 of Example 1A in 0.5 ml water with 200 μl of the stock solution of Example 1B (200 μg of uracil DNA-glycosylase) and incubating at room temperature overnight. HPLC analysis showed quantitative removal of uracil as indicated by a 1:10 ratio between uracil and the abasic dodecamer oligonucleotide. The uracil retention time was 2.43 minutes and the abasic oligonucleotide was 21.68 minutes. The solution was lyophilized and stored in the freezer until further use. 
     D. Preparation of Oligonucleotide Containing Multiple Uridine Sites 
     In the manner of Example 1A the following oligonucleotide was prepared GAC AGA GGU AGG AGA AGU GA (SEQ ID NO: 5) wherein U represents a 2&#39;-deoxyuridine nucleotide. The oligonucleotide is treated in accordance with the procedure of Example 1C resulting in an oligonucleotide of the sequence GAC AGA GGN AGG AGA AGN GA (SEQ ID NO: 6) wherein N represents an abasic site within the oligonucleotide. 
     Example 2 
     Preparation of an Abasic Site Containing Oligonucleotide via a Abasic Sugar Precursor 
     A. Preparation of 5-O-4,4&#39;-Dimethoxytrityl-1,2-Dideoxy-1-(o-nitrobenzoyl)-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N&#39;-Diisopropyl) Phosphoramidite. 
     5-O-4,4&#39;-dimethoxytrityl-1,2-dideoxy-D-ribofuranose-3-O-(2-cyanoethyl-N,N&#39;-diisopropyl) phosphoramidite is prepared in accordance with modification of the procedures of Lyer, et al. Nucleic Acids Research 18: 2855 (1990) and Didier, et al., Tetrahedron Letters 32: 207 (1991) incorporated by reference herein in their entireties. 
     B. Preparation of Oligonucleotide Containing Abasic Site 
     Oligonucleotide having the sequence CGC AGN CAG CC wherein N represents an abasic site (SEQ ID NO:4) from Example 1C can be prepared in accordance with modifications of the procedures of Lyer, et al. Nucleic Acids Research 18: 2855 (1990) and Didier, et al., Tetrahedron Letters 32: 207 (1991). In accordance with these procedures, an o-nitrobenzyl deoxyfuranose containing oligonucleotide is synthesized using the oligonucleotide synthetic methods of Lyer, et al. and Didier, et al. Photolysis utilizing a high intensity Hg lamp (300 nm) generates the corresponding abasic site containing oligonucleotide. Such abasic oligonucleotides are also described in Horn, et al., Nucleosides and Nucleotides 10:299 (1991). 
     Example 3 
     Preparation of Modified Abasic Sugar Precursors 
     A. Preparation of 5-O-(4,4&#39;-Dimethoxytrityl)-2-O-Methyl-1,2-Dideoxy-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N&#39;-Diisopropyl) Phosphoramidite. 
     1-O-methyl-D-ribofuranose is 3,5 protected with TIPS-Cl 2 . It is then 2-position methylated with either diazomethane or methyl iodide/silver oxide (CH 3  I/Ag 2  O). The composition is then treated with an acetic anhydride/acetic acid/sulfuric acid mixture to give a 1-O-acetyl, 2-O-methyl 3,5 protected sugar. The 1-O-acetyl, 2-O-methyl 3,5 protected sugar is deprotected with tetrabutyl ammonium fluoride, 5-position dimethoxytritylated, and 3-position phosphitylated. Thereafter, this phosphoramidite may be incorporated into an oligonucleotide by standard phosphoramidite procedures and ammonia deprotected to form a 2&#39;-O-methyl, 1&#39; abasic site containing oligonucleotide. 
     B. Preparation of 5-O-4,4&#39;-Dimethoxytrityl-2-O-Methyl-1,2-Dideoxy-1-(o-nitrobenzoyl)-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N&#39;-Diisopropyl) Phosphoramidite. 
     1-O-acetyl 2,3,5-tri-O-benzoyl-D-ribofuranose is condensed with o-nitrobenzyl alcohol under Vorbruggen conditions. The resultant 1-O-(ortho-nitrobenzyl)-2,3,5-tri-O-benzoyl (α,δ)-D-ribofuranose is deprotected with ammonia and subsequently treated with TIPS-Cl 2 . The resultant 3,5-silyl protected 1-O-(ortho-nitro benzyl) D-ribofuranose is reacted with diazomethane or CH 3  I/Ag 20  to give the required 2-O-methyl compound. Subsequent3,5-deprotection, 5-dimethoxytritylation and 3-phosphitylation gives the named phosphoramidite. The phosphoramidite can be incorporated into an oligonucleotide via standard phosphoramidite procedures. 
     C. Preparation of 5-O-(4,4&#39;-Dimethoxytrityl)-2-Fluoro-1,2-Dideoxy-D-Ribofuranose-3-O-(2-Cyanoethyl-N,N&#39;-Diisopropyl) Phosphoramidite. 
     1-O-(ortho-nitrobenzyl)-2,3,5-tri-O-benzoyl-D-ribofuranose is deprotected at 2,3,5 positions using ammonia. Tritylation with excess trityl chloride/pyridine/4-dimethylaminopyridine gives 3-5-ditrityl-1-O-nitrobenzyl-D-ribo furanose. Oxidation at 2 position with CrO 3  followed by NaBH 4  reduction inverts the configuration at 2 position yielding an arabino sugar. The arabino sugar is converted to its triflate at 2 position and the triflate is displaced with fluoride ion to yield the 2-fluoride modified sugar which can be 5 position protected and phosphitylated to incorporate the sugar into an oligonucleotide via standard oligonucleotide synthesis. 
     Example 4 
     Oligonucleotides conjugated in the following example are set forth in Table 2. 
     
                       TABLE II                                                    
______________________________________                                    
OLIGOMER                                                                  
(SEQ ID NO.)                                                              
         TARGET   SEQUENCE     LINKER (L)                                 
______________________________________                                    
A (SEQ ID                                                                 
         ICAM     TGG GAG CCA  3-carbon                                   
                                       P = S                              
NO: 7)            TAG CGA GGC-L                                           
                               amino                                      
B (SEQ ID                                                                 
         ICAM     TGG GAG CCA  3-carbon                                   
                                       P = O                              
NO: 7)            TAG CGA GGC-L                                           
                               amino                                      
C (SEQ ID                                                                 
         BPV      CTG TCT CCA* 2&#39;amino-                                   
                                       P = O                              
NO: 8)            TCC TCT TCA CT                                          
                               pentoxy                                    
D (SEQ ID                                                                 
         BPV      CTG TCT CCA  3-carbon                                   
                                       P = O                              
NO: 9)            TCC TCT TCA  amino                                      
                  CT-L                                                    
E (SEQ ID                                                                 
         BPV      CTG TCT CCA  6-carbon                                   
                                       P = O                              
NO: 9)            TCC TCT TCA  amino                                      
                  CT-L                                                    
F (SEQ ID                                                                 
         CMV      GGC GUC UCC          2&#39;-OMe                             
NO: 10)           AGG CGA UCU                                             
                  GAC*                                                    
G (SEQ ID                                                                 
         ICAM     TCT GAG TAG          2&#39;-OMe                             
NO: 11)           CAG AGG AGC                                             
                  TC*                                                     
H (SEQ ID         GGA UGG CGU          2&#39;-OMe                             
NO: 12)           CUC CAG GCG                                             
                  AUC*                                                    
I (SEQ ID         GGA UGG CGU  3-carbon                                   
                                       2&#39;-OMe                             
NO: 13)           CUC CAG GCG  amino                                      
                  AUC-L                                                   
J (SEQ ID         GGA UGG CGU  6-carbon                                   
                                       2&#39;-OMe                             
NO: 13)           CUC CAG GCG  amino                                      
                  AUC-L                                                   
K (SEQ ID         F-TGG GAG CCA                                           
                               3-carbon                                   
                                       2&#39;-OMe                             
NO: 7)            TAG CGA GGC-L                                           
                               amino                                      
______________________________________                                    
 A* = 2O-aminopentoxy-2deoxyadenosine                                     
 C* = 2aminopropoxy cytosine                                              
 F = Fluoroscein                                                          
 
    
     A. 3&#39; Terminus Polyamine End Labeled Oligonucleotide 
     1. 3&#39;-Terminus Polyamine Oligonucleotide I 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester oligonucleotide having the sequence D-polyamine  (SEQ ID NO: 9)-polyamine!, wherein the polyamine is one of the following: 
     
                       TABLE III                                                   
______________________________________                                    
1,6 Diaminohexane    Oligomer D (i)                                       
Diethylenetriamine   Oligomer D (ii)                                      
Triethylenetetramine Oligomer D (iii)                                     
Spermine             Oligomer D (iv)                                      
Pentaethylenehexamine                                                     
                     Oligomer D (v)                                       
______________________________________                                    
 
    
     a. Preparation of the Intermediate Linker 
     The oligonucleotide sequence having a 3&#39;-terminus amino group was synthesized using 3&#39;-amino modifier (with a three carbon linker) controlled pore glass (CPG) from Glen Research as the solid support. The synthesis was conducted with an Applied Biosystems 380B or 994 in the &#34;Trityl-Off&#34; mode. The resultant oligonucleotide was cleaved from the solid support and deprotected with concentrated NH 4  OH for 16 hrs at 55° C. Purification on a Sephadex G-25 column yielded a 3&#39;-amino modified oligonucleotide of the specified sequence. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The crude 3&#39;-aminolinker-oligonucleotide (SEQ ID NO:9) (15 O.D. units, approximately 85 nmols) was dissolved in freshly prepared NaHCO 3  buffer (150 ul, 0.2M, pH 8.1) and treated with a solution of disuccinimidyl suberate (DSS) (approximately 5 mgs) dissolved in 150 ul of methyl sulfoxide (DMSO). The reaction mixture was left to react for 20 minutes at room temperature. The mixture was then passed over a Sephadex G-25 column (0.7×45 cm) to separate the activated oligonucleotide-DSS from the excess DSS. The oligonucleotide-DSS was then frozen immediately and lyophilized to dryness. A solution of polyamine in 0.33M NaOAc (approximately 6 mg polyamine in 300 ul 0.33M NaOAc, pH 5.2, final solution pH 6-8.0) was added to the dried oligonucleotide-DSS, and this mixture was allowed to react overnight at room temperature. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using a Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 4. 
     
                       TABLE IV                                                    
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted D    26.44 mins                                                 
Oligomer D (i) 27.48 mins                                                 
Oligomer D (ii)                                                           
               27.23 mins                                                 
Oligomer D (iii)                                                          
               27.27 mins                                                 
Oligomer D (iv)                                                           
               27.54 mins                                                 
Oligomer D (v) 27.36 mins                                                 
______________________________________                                    
 
    
     In a second test run under the same conditions the HPLC gradient was 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 15% solvent B in 60 minutes. HPLC retention times were as set forth in Table 5. 
     
                       TABLE V                                                     
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreated D     60.74 mins                                                 
Oligomer D (ii)                                                           
               62.37 mins                                                 
Oligomer D (v) 65.24 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugates (the larger the polyamine, the slower the migration) versus the oligonucleotide alone. (Gel: 313-107) 
     c. Nuclease stability of 3&#39; polyamine conjugates in Fetal Calf Serum 
     Polyamine conjugates of the invention are assessed for their resistance to serum nucleases by incubation of the oligonucleotides in media containing various concentrations of fetal calf serum. Labeled oligonucleotides are incubated for various times, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamide-urea denaturing gels and subsequent autoradiography or phosphor-imaging. Autoradiograms are quantitated by laser densitometry. Based upon the location of the modifications and the known length of the oligonucleotide it is possible to determine the effect of the particular modification on nuclease degradation. For the cytoplasmic nucleases, a HL60 cell line is used. A post-mitochondrial supernatant is prepared by differential centrifugation and the labeled oligonucleotides are incubated in this supernatant for various times. Following the incubation, oligonucleotides are assessed for degradation as outlined above for serum nucleolytic degradation. Autoradiography results are quantitated for comparison of the unmodified and the modified oligonucleotides. The t 1/2   are set forth below. 
     
                       TABLE VI                                                    
______________________________________                                    
Oligonucleotide    t.sub.1/2 (hours)                                      
______________________________________                                    
wild type oligomer D                                                      
                   0.5 (no aminolinker)                                   
unreacted oligomer D                                                      
                   22 (with aminolinker)                                  
oligomer D (ii)    48                                                     
oligomer D (v)     &gt;50                                                    
______________________________________                                    
 
    
     2. 3&#39;-Terminus Polyamine Conjugate II 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester oligonucleotide having the sequence E-polyamine  (SEQ ID NO: 9)-polyamine! wherein the polyamine is one of the following: 
     
                       TABLE VII                                                   
______________________________________                                    
Diethylenetriamine    Oligomer E (i)                                      
Pentaethylenehexamine Oligomer E (ii)                                     
______________________________________                                    
 
    
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared as described in Example 4-A-1-a substituting a 3&#39; amino modifier with a six carbon linker (Clonetech, Palo Alto, Calif.) for the 3&#39;-amino modifier (with a three carbon linker. 
     3. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide was prepared in accordance with Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 25% solvent B in the next 50 minutes using a Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 8. 
     
                       TABLE VIII                                                  
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreated E     41.38 mins                                                 
Oligomer E (i) 43.29 mins                                                 
Oligomer E (ii)                                                           
               43.43 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugates (the larger the polyamine, the slower the migration) versus the oligonucleotide alone. (Gel: 353-35). 
     4. 3&#39; -Terminus Polyamine Conjugate III 
     Polyamines were attached to the 3&#39;-terminus end of a phosphorothioate oligonucleotide having the sequence A-polyamine  (SEQ ID NO:7)-polyamine! where the polyamine is one of the following: 
     
                       TABLE IX                                                    
______________________________________                                    
1,6 Diaminohexane    Oligomer A (i)                                       
Diethylenetriamine   Oligomer A (ii)                                      
Triethylenetetramine Oligomer A (iii)                                     
Spermine             Oligomer A (iv)                                      
Pentaethylenehexamine                                                     
                     Oligomer A (v)                                       
______________________________________                                    
 
    
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared as described in Example 4-A-1-a utilizing the Beaucage reagent (3H-1,2-benzodithioate-3-one 1,1-dioxide, see Radhakrishnan, P. I., Egan, W., Regan, J. B. and Beaucage, S. L., (1990) J. Am. Chem. Soc., 112:1253) to form the phosphothioate internucleotide backbone. The 3&#39;-aminolinker was introduced as described in example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     Oligonucleotides were functionalized as described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using a Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 10. 
     
                       TABLE X                                                     
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted A    30.77 mins                                                 
Oligomer A (iii)                                                          
               31.31 mins                                                 
Oligomer A (v) 31.02 mins                                                 
______________________________________                                    
 
    
     In a second test run under the same conditions, the HPLC gradient was 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 15% solvent B in 60 minutes. Retention times were as set forth in Table 11. 
     
                       TABLE XI                                                    
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreated A     68.62 mins                                                 
Oligomer A (i) 68.70 mins                                                 
Oligomer A (ii)                                                           
               68.69 mins                                                 
______________________________________                                    
 
    
     In a second test run under the same conditions, HPLC retention times were as set forth in Table 12. 
     
                       TABLE XII                                                   
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreated A     30.34 mins                                                 
Oligomer A (iv)                                                           
               30.57 mins                                                 
Oligomer A (v) 30.72 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugates (the larger the polyamine, the slower the migration) versus the oligonucleotide alone. (Test run 1 Gel, 313-82; Test run 2 Gel, 285-138; Test run 3 Gel, 353-57) 
     C. Preparation of Biotin Functionalized Oligonucleotide Polyamine Conjugate 
     To further characterize the oligonucleotide polyamine conjugate, biotin was attached to the free amines made available by the polyamines attached in Example 4-A-4-b. About 10 O.D. units (A 260 ) of Oligomers A(i) and A(ii) (approximately 58 nmoles) were dried in a microfuge tube. The oligonucleotide polyamine conjugate was rehydrated in 400 ul of 0.2M NaHCO 3  (pH 8.1) buffer and D-biotin-N-hydroxysuccinimide ester (approximately 5.0 mgs biotin for the 1,6 Diaminohexane conjugate, 8.0 mgs for the Diethylenetriamine) (Sigma) was added followed by 200 ul of DMF. The solution was left to react overnight at room temperature. The solution was then passed over a NAP-25 column and analyzed by reverse phase HPLC. Solvent A was 50 mM TEAA and solvent B was CH 3  CN. The HPLC gradient was 0-10 mins, 95% A, 5% B; linear increase to 40% B in the next 50 minutes using a Water&#39;s Delta-Pak C-18, reverse phase column. The HPLC retention times were as set forth in Table 13. 
     
                       TABLE XIII                                                  
______________________________________                                    
Oligomer         Retention Time                                           
______________________________________                                    
unreated A       30.77 mins                                               
Oligomer A (i)   31.31 mins                                               
Oligomer A (i)-Biotin                                                     
                 35.56 mins                                               
Oligomer A (ii)  31.02 mins                                               
Oligomer A (ii)-Biotin                                                    
                 36.23 mins                                               
______________________________________                                    
 
    
     5. 3&#39; -Terminus Polyamine Conjugate IV 
     Polyamines were attached to the 3&#39;-terminus end of the phosphodiester oligonucleotide having the sequence B-polyamine  (SEQ ID NO: 7)-polyamine! wherein the polyamine is one of the following: 
     
                       TABLE XIV                                                   
______________________________________                                    
Diethylenetriamine   Oligomer B (i)                                       
Triethylenetetramine Oligomer B (ii)                                      
Spermine             Oligomer B (iii)                                     
Pentaethylenehexamine                                                     
                     Oligomer B (iv)                                      
______________________________________                                    
 
    
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared as described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The oligonucleotide was functionalized with polyamines as described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using a Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 15. 
     
                       TABLE XV                                                    
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
untreated B    25.71 mins                                                 
Oligomer B (i) 26.11 mins                                                 
Oligomer B (ii)                                                           
               25.26 mins                                                 
Oligomer B (iii)                                                          
               25.10 mins                                                 
Oligomer B (iv)                                                           
               25.12 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugates (the larger the polyamine, the slower the migration) versus the oligonucleotide alone. (Gel: 313-112) 
     B. 2&#39; Internal Polyamine Labeled Oligonucleotide 
     1. 2&#39;-Internal Polyamine oligonucleotide I 
     Polyamines were attached to the 2&#39;-internal linker site of a phosphodiester oligonucleotide having the sequence C-polyamine  (SEQ ID NO: 8)-polyamine! wherein the polyamine is one of the following: 
     
                       TABLE XVI                                                   
______________________________________                                    
Diethylenetriamine   Oligomer C (i)                                       
Triethylenetetramine Oligomer C (ii)                                      
Pentaethylenehexamine                                                     
                     Oligomer C (iii)                                     
______________________________________                                    
 
    
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared as described in Example 4-A-1-a incorporating a modified adenosine phosphoramidite (with a 2&#39;-aminolinker) at position #9. This oligonucleotide and the 2&#39;-amino linker have been described in Manoharan, M., Guinosso, C. J., Cook, P. D., Tetrahedron Letters, 32, (pgs. 7171-7174), 1991. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The oligonucleotide was functionalized as described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using a Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 17. 
     
                       TABLE XVII                                                  
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
untreated C    26.20 mins                                                 
Oligomer C (i) 27.52 mins                                                 
Oligomer C (ii)                                                           
               27.50 mins                                                 
Oligomer C (iii)                                                          
               27.59 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugates (the larger the polyamine, the slower the migration) versus the oligonucleotide alone. (Gel: 313-97) 
     C. 3&#39; Terminus Polyamine End Labeled Oligonucleotide, Using a 2&#39;-aminolinker 
     1. 3&#39; Terminus Polyamine Labeled Oligonucleotide I, Using a 2&#39;-aminolinker 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide via a 2&#39;-aminolinker having the sequence F-polyamine  (SEQ ID NO:10)-polyamine! wherein the polyamine is pentaethylenehexamine (oligomer F(i)). 
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared as described in Example 4-A-1-a, except that a modified cytosine CPG (with a 2&#39;-propylaminolinker) was introduced at the 3&#39; end. The 2&#39;-modification can be prepared by modification of the procedure previously described in application Ser. No. 918,362 filed Jul. 23, 1992. The CPG containing 2&#39;-w-phthalimido-propoxy-cytidine was synthesized according to the standard protocols reported in the literature. See, for example, B. S. Sproat and A. I. Lamond, in &#34;Oligonucleotides and Analogues&#34; edited by F. Eckstein , IRL Press at Oxford University Press (1991) p71-72. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide was prepared in accordance with Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 18. 
     
                       TABLE XVIII                                                 
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted F    28.53 mins                                                 
oligomer F (i) 29.47 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 397-85) 
     2. 3&#39; Terminus Polyamine Labeled Oligonucleotide II, Using a 2&#39;-aminolinker 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide via a 2&#39;-aminolinker having the sequence G-polyamine  (SEQ ID NO:11)-polyamine! wherein the polyamine is pentaethylenehexamine (oligomer G(i)). 
     a. Preparation of the Intermediate Linker 
     The intermediate linker was prepared in accordance with the method described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide was prepared in accordance with the procedures described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 19. 
     
                       TABLE XIX                                                   
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted G    28.43 mins                                                 
oligomer G (i) 29.06 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 397-85) 
     3. 3&#39; Terminus Polyamine Labeled Oligonucleotide III Using a 2&#39;-aminolinker 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide via a 2&#39;-aminolinker having the sequence H-polyamine  (SEQ ID NO:12)-polyamine! wherein the polyamine is pentaethylenehexamine. 
     a. Preparation of the Intermediate Linker 
     The intermediate linker is prepared in accordance with methods described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide is prepared in accordance with methods described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 20. 
     
                       TABLE XX                                                    
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted H    28.49 mins                                                 
oligomer H (i) 30.36 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 397-85) 
     Example 5 
     Polyamine Labeled 2&#39;-OMe Oligonucleotides and Other RNA Mimics 
     1. Polyamine Labeled 2&#39;-OMe Oligonucleotide I 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide (via a 3 carbon linker) having the sequence I-polyamine  (SEQ ID NO:13)-polyamine! wherein the polyamine is pentaethylenehexamine (oligomer I(i)). 
     a. Preparation of the Intermediate Linker 
     The intermediate linker is prepared in accordance with methods described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide is prepared in accordance with methods described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 21. 
     
                       TABLE XXI                                                   
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted I    28.93 mins                                                 
oligomer I (i) 29.59 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 353-156) 
     2. Polyamine Labeled 2&#39;-OMe Oligonucleotide II 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide (via a 6 carbon linker) having the sequence J-polyamine  (SEQ ID NO:13)-polyamine! wherein the polyamine is pentaethylenehexamine (oligomer J(i)). 
     a. Preparation of the Intermediate Linker 
     The intermediate linker is prepared in accordance with methods described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide is prepared in accordance with methods described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 22. 
     
                       TABLE XXII                                                  
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted J    28.76 mins                                                 
oligomer J (i) 29.39 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 397-85) 
     3. Polyamine Labeled 2&#39;-OMe oligonucleotide III 
     Polyamines were attached to the 3&#39;-terminus end of a phosphodiester (2&#39;-OMe) oligonucleotide (via a 3 carbon linker) having another reporter group (such as biotin, fluorescein) at the other end in the sequence K-polyamine  (SEQ ID NO:7)-polyamine!. Fluoroscein at 5&#39; end was added using the required amidite commercially available from Clontech. The polyamine is one of the following 
     pentaethylenehexamine oligomer K(i) 
     spermine oligomer K(ii) 
     a. Preparation of the Intermediate Linker 
     The intermediate linker is prepared in accordance with methods described in Example 4-A-1-a. 
     b. Preparation of Polyamine Functionalized Oligonucleotide 
     The polyamine functionalized oligonucleotide is prepared in accordance with methods described in Example 4-A-1-b. The resulting polyamine-oligonucleotide conjugate was characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 23. 
     
                       TABLE XXIII                                                 
______________________________________                                    
Oligomer       Retention Time                                             
______________________________________                                    
unreacted K    31.35 mins                                                 
oligomer K (i) 31.96 mins                                                 
oligomer K (ii)                                                           
               32.15 mins                                                 
______________________________________                                    
 
    
     Gel analysis showed progressively slower migration times for the polyamine conjugate versus the oligonucleotide alone. (Gel: 353-149) 
     Example 6 
     5&#39; Terminus Polyamine End Labeled Oligonucleotide 
     1. 5&#39;-Terminus Polyamine Oligonucleotide I 
     Polyamines were attached to the 5&#39;-terminus end of a phosphodiester oligonucleotide having the following sequences: 
     5&#39;-aminolinker-TCAG (oligomer L) 
     5&#39;-aminolinker-CGCACGC (oligomer M) to provide the polyamine oligonucleotides: 
     5&#39;-polyamine-TCAG (oligomer L(i)) 
     5&#39;-polyamine-CGCACGC (oligomer M(i)) wherein the polyamine is pentaethylenehexamine. 
     a. Preparation of the Intermediate Linker 
     The oligonucleotide sequence having a 5&#39;-terminus amino group was synthesized using Aminolink-II(with a six carbon linker) phosphoramidite from Applied Biosystems in the last round of synthesis. The synthesis was conducted with an Applied Biosystems 380B or 994 in the &#34;Trityl-On&#34; mode. The resultant oligonucleotide was cleaved from the solid support and deprotected with concentrated NH 4  OH for 16 hrs at 55° C. Purification on a Sephadex G-25 column yielded a 5&#39;-amino modified oligonucleotide of the specified sequence. 
     b. Preparation of Polyamine Functionalized Oligonucleotide L(i) 
     The crude 5&#39;-aminolinker-oligonucleotide (150 O.D. units, approximately 3.75 mmols) was dissolved in freshly prepared NaHCO 3  buffer (900 ul, 0.2M, pH 8.1) and treated with a solution of disuccinimidyl suberate (DSS) (approximately 30 mgs) dissolved in 750 ul of methyl sulfoxide (DMSO). The reaction mixture was left to react for 20 minutes at room temperature. The mixture was divided into three portions and then passed over a Sephadex G-25 column (0.7×45 cmx3columns) to separate the activated oligonucleotide-DSS from the excess DSS. The oligonucleotide-DSS was then frozen immediately and lyophilized to dryness. A solution of polyamine in 0.33M NaOAc (approximately 60 mL polyamine in 1950 ul 0.33M NaOAc, pH 5.2, final solution pH 6-8.0) was added to the dried oligonucleotide-DSS, and this mixture was allowed to react overnight at room temperature. 
     c. Preparation of Polyamine Functionalized Oligonucleotide M(i) 
     The crude 5&#39;-aminolinker-oligonucleotide (oligomer M) (150 O.D. units, approximately 2.50) was reacted as described in Example 6(b). 
     d. Characterization of 5&#39; Polyamine Functionalized Oligonucleotides 
     The resulting polyamine-oligonucleotide conjugates were characterized by reverse phase HPLC and a 20% denaturing gel. Solvent A was 50 mM TEAA, solvent B was CH 3  CN. The HPLC gradient was from 0-10 mins, 95% solvent A, 5% solvent B; linear increase to 40% solvent B in the next 50 minutes using Water&#39;s Delta-Pak C-18 reverse phase column. HPLC retention times were as set forth in Table 24. 
     
                       TABLE XXIV                                                  
______________________________________                                    
        DNA TARGET   RNA TARGET                                           
          T.sub.m                                                         
                 ΔT.sub.m                                           
                        ΔΔG.sup.o.sub.37°C.            
                               (°C.)                               
                                    (°C.)                          
                                         ΔΔG.sup.o.sub.37.degr
                                         ee.C.                            
______________________________________                                    
Oligomer  (°C.)                                                    
                 (°C.)                                             
                        ΔΔG.sup.o.sub.37°C.            
                               (°C.)                               
                                    (°C.)                          
                                         ΔΔG.sup.o.sub.37.degr
                                         ee.C.                            
______________________________________                                    
wild type 60.6   --     --     64.9 --   --                               
oligomer D                                                                
oligomer D                                                                
          60.3   -0.3   +0.3   64.6 -0.3 0.0                              
oligomer D +                                                              
          60.8   +0.2   0.0    65.1 +0.2 0.0                              
5&#39;-6-carbon                                                               
amino linker)                                                             
oligomer E                                                                
          60.8   +0.2   -0.8   65.8 +0.9 -1.0                             
oligomer E(i)                                                             
          61.2   +0.6   -1.4   66.3 +1.4 -1.9                             
oligomer E +                                                              
          61.5   +0.9   -1.7   67.1 +2.2 -2.1                             
spermine                                                                  
oligomer E(ii)                                                            
          61.2   +0.6   -1.3   67.5 +2.6 -2.6                             
______________________________________                                    
 
    
     Example 7 
     Preparation of a Reactive Site containing Oligonucleotide 
     An oligonucleotide having the sequence TGGGAGCCATAGCGAGGUCT (SEQ ID NO: 14) is treated with uracil DNA glycosylase followed by T4 endonuclease. The product is then treated with 1-phthalimidobutyl-4-thiol. Nucleophilic attack by the thiol adds to the protected aminobutyl moiety to what was the 3&#39; position of the opened nucleotide. Treatment of this composition with hydrazine will deblock the phthalimide yielding an amino species which is then treated with bifunctional linker followed by treatment with an appropriate polyamine species as per Example 4-A-1-b. 
     Example 8 
     Preparation of Polyamine Conjugated Oligonucleotide 
     An oligonucleotide is prepared as described in Example 7 treating the product with NH 2  --CH 2  --CH 2  --SH. The thiol group will attack the double bond of the opened nucleotide. This species may then be further derivatized with a reactive group. 
     Example 9 
     Thermodynamic Parameters of Oligoamine-Oligonucleotide Conjugates with DNA and RNA Targets 
     The ability of the functionalized oligonucleotides of the invention to hybridize to their complementary RNA or DNA sequences is determined by thermal melting analysis. The RNA complement is synthesized from T7 RNA polymerase and a template-promoter of DNA synthesized with an Applied Biosystems, Inc. 380B nucleic acid synthesizer. The RNA species is purified by ion exchange using FPLC (LKB Pharmacia, Inc.) or by denaturing urea-PAGE. Natural antisense oligonucleotides or those containing functionalization at specific locations are added to either the RNA or DNA complement at stoichiometric concentrations to form hybrid duplexes. The absorbance (260 nm) hyperchromicity dependence on temperature upon duplex to random coil transition is monitored using a Gilford Response II spectrophotometer. These measurements are performed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and NaCl to yield an ionic strength of either 0.1M or 1.0M. Data are analyzed by a graphic representation of 1/T m  vs ln Ct!, where  Ct! is the total oligonucleotide concentration. From this analysis the thermodynamic parameters are determined. Based upon the information gained concerning the stability of the duplex or hetero-duplex formed, the placement of the polyamines into oligonucleotides is assessed for its effects on helix stability. Modifications that drastically alter the stability of the hybrid exhibit reductions or enhancements in the free energy (delta G) and decisions concerning their usefulness in antisense oligonucleotides are made. 
     Example 10 
     Conjugation of Polyamines to Abasics Sites Containing Oligonucleotides 
     To 15.2 ODS of an abasic oligonucleotide (SEQ ID NO: 4) in 100 μl water was added 25 μl 1M NaOAc (pH 5.0) solution. The final concentration of the acetate buffer was 0.2M. 5.3 mg of triethylenetetramine was dissolved in 500 μl of 1M NaOAc (pH 5.0) solution. 50 μl of the resulting solution was added to the oligonucleotide solution followed by 50 μl of NaCNBH 3  (57 MM solution). The pH of the resulting solution was below 8.0. The solution was vortexed and left to stand overnight. HPLC and Gel analysis indicated conjugation of the triethylenetetramine to the oligonucleotide. The conjugated oligonucleotide was purified by G-25 and HPLC. HPLC retention times are set forth in Table 25. 
     
                       TABLE XXV                                                   
______________________________________                                    
Oligomer              Retention time (mins)                               
______________________________________                                    
parent oligonucleotide (SEQ ID NO: 3)                                     
                      26.66                                               
abasic oligonucleotide (SEQ ID NO: 4)                                     
                      26.16                                               
(SEQ ID NO: 4)-triethylenetetramine conjugate                             
                      26.04                                               
______________________________________                                    
 
    
     
         __________________________________________________________________________
SEQUENCE LISTING                                                          
(1) GENERAL INFORMATION:                                                  
(iii) NUMBER OF SEQUENCES: 14                                             
(2) INFORMATION FOR SEQ ID NO:1:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 18 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                   
TGGGAGCCATAGCGAGGC18                                                      
(2) INFORMATION FOR SEQ ID NO:2:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 19 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 19                                                          
(D) OTHER INFORMATION: /note= &#34;abasic, aldehydic                          
species&#34;                                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                   
TGGGAGCCATAGCGAGGCN19                                                     
(2) INFORMATION FOR SEQ ID NO:3:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 11 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 6                                                           
(D) OTHER INFORMATION: /note= &#34;2&#39;deoxyuridine                             
residue&#34;                                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                   
CGCAGUCAGCC11                                                             
(2) INFORMATION FOR SEQ ID NO:4:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 11 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 6                                                           
(D) OTHER INFORMATION: /note= &#34;abasic residue&#34;                            
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                   
CGCAGNCAGCC11                                                             
(2) INFORMATION FOR SEQ ID NO:5:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 9                                                           
(D) OTHER INFORMATION: /note= &#34;2&#39;deoxyuridine                             
residue&#34;                                                                  
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 18                                                          
(D) OTHER INFORMATION: /note= &#34;2&#39;deoxyuridine                             
residue&#34;                                                                  
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                   
GACAGAGGUAGGAGAAGUGA20                                                    
(2) INFORMATION FOR SEQ ID NO:6:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 9                                                           
(D) OTHER INFORMATION: /note= &#34;abasic residue&#34;                            
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 18                                                          
(D) OTHER INFORMATION: /note= &#34;abasic residue&#34;                            
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                   
GACAGAGGNAGGAGAAGNGA20                                                    
(2) INFORMATION FOR SEQ ID NO:7:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 18 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                   
TGGGAGCCATAGCGAGGC18                                                      
(2) INFORMATION FOR SEQ ID NO:8:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 9                                                           
(D) OTHER INFORMATION: /note=                                             
&#34;2&#39;-O-aminopentoxy-2&#39;-deoxyadenosine&#34;                                     
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                   
CTGTCTCCATCCTCTTCACT20                                                    
(2) INFORMATION FOR SEQ ID NO:9:                                          
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                   
CTGTCTCCATCCTCTTCACT20                                                    
(2) INFORMATION FOR SEQ ID NO:10:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 21 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 21                                                          
(D) OTHER INFORMATION: /note= &#34;2&#39;-aminopropoxy                            
cytosine&#34;                                                                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                  
GGCGUCUCCAGGCGAUCUGAC21                                                   
(2) INFORMATION FOR SEQ ID NO:11:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 20                                                          
(D) OTHER INFORMATION: /note= &#34;2&#39;-aminopropoxy                            
cytosine&#34;                                                                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                  
TCTGAGTAGCAGAGGAGCTC20                                                    
(2) INFORMATION FOR SEQ ID NO:12:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 21 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(ix) FEATURE:                                                             
(A) NAME/KEY: misc.sub.-- feature                                         
(B) LOCATION: 21                                                          
(D) OTHER INFORMATION: /note= &#34;2&#39;-aminopropoxy                            
cytosine&#34;                                                                 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                  
GGAUGGCGUCUCCAGGCGAUC21                                                   
(2) INFORMATION FOR SEQ ID NO:13:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 21 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                  
GGAUGGCGUCUCCAGGCGAUC21                                                   
(2) INFORMATION FOR SEQ ID NO:14:                                         
(i) SEQUENCE CHARACTERISTICS:                                             
(A) LENGTH: 20 base pairs                                                 
(B) TYPE: nucleic acid                                                    
(C) STRANDEDNESS: single                                                  
(D) TOPOLOGY: linear                                                      
(ii) MOLECULE TYPE: DNA (genomic)                                         
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                  
TGGGAGCCATAGCGAGGUCT20                                                    
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