Synthetic oligonucleotides have been shown to regulate gene expression in cells. Thus, these molecules bind to target regions of nucleic acids and inhibit gene expression. They are useful to control infectious disease and have been used and tested in a variety of diseases or conditions associated with altered or aberrant gene expression such as cancers, hereditary disorders, and the like.
There are two major oligonucleotide (ODN) based strategies designed to inhibit gene expression. One method uses antisense ODNs complementary to a specific mRNA to form DNA:RNA hybrids. These hybrids are stabilized by Watson-Crick base pairing and are substrates for cellular ribonuclease H (RNase H), an enzyme that degrades the RNA portion of the duplex rendering the mRNA untranslatable (Walder, et al., Proc. Natl. Acad. Sci. USA, 85:5011-5015, 1988). Because RNase H does not degrade the ODN, the ODN is able to hybridize to another copy of the target mRNA. Modified oligonucleotides have been disclosed for duplex-forming oligonucleotides and in one example, Letsinger et al. disclose the use of N,N,N'-trimethylethylenediamine or 4-(2 aminoethyl)morpholine internucleoside linkages to promote duplex formation (J. Am. Chem. Soc. 110;4470-4471, 1988)
A second ODN-based strategy for altering gene expression targets DNA by the formation of a triple helical structure. The use of ODNs to form triple helix structures was initially reported by Moser, et al. (Science, 238:645-650, 1987, and see LeDoan, et al., Nucl. Acids Res., 15:7749-7760, 1987). Under suitable conditions, an ODN will bind in the major groove of a DNA duplex. The presence of a third strand may either sterically block transcription, prevent the sequence specific interactions of regulatory proteins with DNA, and/or alter the conformation of the bound duplex.
There are two known triplex binding motifs, both involving interactions between the bases of a relatively short ODN (generally between 11-50 base pairs) and the purine bases of a polypurine:polypyrimidine stretch of duplex DNA. In the pyrimidine motif, thymidine residues in the third strand interact with adenosine residues of an A:T duplex while a protonated cytidine in the third strand is hydrogen-bonded to the guanosine of a G:C duplex (Moser, et al., supra). The protonation of C residues at nitrogen position number three generally requires a pH less than six, and thus limits the use of this strategy in vivo (Rajagopal, P., et al., Nature, 339:637-640, 1989, and Lyamichev, et al., Nucl. Acids Res., 16:2165-2178, 1988) unless deoxycytidine analogues are used (see Krawczyk, et al., Proc. Natl. Acad. Sci. (ISA), 89:3761-3764, 1992).
The second triplex motif involves a purine rich triplex forming oligonucleotide (TFO). Thymidine or adenosine residues of the third strand bind to the adenosine of an A:T duplex and guanosine in the third strand interacts with the guanosine of a G:C duplex (Beal, et al., Science, 251:1360-1363, 1991, Durland, et al., Biochem., 30:9246-9255, 1955, and Cooney, et al., Science, 241:456-459, 1988). The orientation of the third strand has been shown to be antiparallel to the purine-rich strand of the duplex (Beal, et al., supra). The major drawback to using this approach in vivo is the tendency of G-rich ODNs to self-associate into quartets at physiologic potassium concentrations. However, recent studies indicate that the use of a GT rich ODN to affect transcription of a transfected CAT plasmid in vivo indicates that GT rich combinations may minimize quartet formation. Unmodified negatively charged oligonucleotides do not generally stably participate in triplex formation in cells. Triplex forming oligonucleotide strategies must account for the physiologic concentrations of Mg.sup.+2 and the level of potassium tolerated for stable triplex formation.
To improve the stability and cellular uptake of oligonucleotides, oligonucleotides have been prepared having modifications to the phosphate backbone. For example, phosphorothiate and methylphosphonate derivatives of oligonucleotides have been synthesized and have sequence specificity and hybridize similarly to that of unmodified oligonucleotides. Aminoethyl phosphonate derivatives of oligonucleotides have also been synthesized and demonstrate enhanced stability in aqueous solution as compared with aminoethyl phosphonate linkages (Fathi, et al., Nucl. Acids Res., 22:5416-5424, 1994). The uncharged character of the methylphosphonate derivatives permits an enhanced uptake of the oligonucleotides by the cell and increased resistance to nucleases as compared with unmodified oligonucleotides. Methoxyethyl-phosphoramidate linkages have been incorporated into the internucleoside backbone at 3' and 5' linkages to inhibit exonuclease degradation. The synthesis of ODNs with some modified and some unmodified linkages allowed both increased nuclease resistance while maintaining RNase H mediated target RNA degradation. (Dagle, et al., Antisense Res. and Devel., 1:11-20, 1991).
Intercalating agents have also been conjugated to oligonucleotides to increase the stability of the conjugate with the complementary strand (see, for example, Wilson, et al., Biochem., 32:10614-10621, 1993, and Orson, et al., Nucl. Acids Res., 22:479-484, 1994). In addition, molecules are often attached to the oligonucleotides to modify the net charge. Examples of these agents include polylysine, cationic peptides, polyamines and polycationic polymers. Intercalators and polylysine have shown an increased resistance to nuclease degradation.
Nucleomonomers can also be modified to improve triplex formation and PCT International Publication No. WO 94/24144 discloses oligomers with 7-deaza-7-substituted purines.
The formation of a DNA triplex using a purine rich ODN is inhibited by monovalent cations, particularly potassium ions (Olivas, et al., Biochem., 34:278-284, 1995). Intracellular K.sup.+ concentrations inhibit triplex formation using unmodified oligonucleotides. Potassium ions are the predominant intracellular cations. One problem with forming triplexes at physiologic K.sup.+ levels is the self-association of guanosine-rich ODNs into aggregates which are stabilized by guanine quartets (Olivas, et al., Biochem., 34:278-284, 1995; Olivas, et al., Nucl. Acids Res., 23:1936-1941, 1995). In addition to decreasing the rate of triplex association, K.sup.+ increases the rate of triplex disassociation in vitro (Olivas, Biochem., supra). The inhibitory effect of K.sup.+ can be partially diminished by chemical modification of ODNs. For example, incorporation of the modified base 6-thioguanine in place of native guanine into TFOs decreases the association of ODNs into quartets and increases triplex formation in the presence of monovalent cations (Olivas, Nucl. Acids Res., supra, and Rao, et al., Biochem., 34:765-772, 1995).
Another hurdle associated with the use of ODNs in vivo is that the oligonucleotides are rapidly degraded by intracellular nucleases (Rebagliate, et al., Cell, 48:599-605, 1987; Dagle et al., Nucl. Acids Res., 18:4751-4757, 1990; Dagle, et al., Antisense Res. and Dev., 1:11-20, 1991). The chemical modification of ODNs provides resistance to nucleolytic degradation (Dagle, et al., supra), potentially increasing the overall activity of these compounds in vivo (Dagle, et al., supra, and Weeks, et al., Development, 111:1173-1178, 1991). The type and degree of chemical modification of ODNs, however, is limited when strategies require the action of cellular RNase H (Dagle, et al., Antisense, supra). Additionally, some modification of ODNs can result in nonspecific toxicity mediated through non-nucleic acid interactions, such as has recently been reported for phosphorothioate ODNs and the basic fibroblast growth factor receptor (Guvakova, et al., J. Biol. Chem., 270:2620-2627, 1995). In contrast, the formation of triplex structures does not require an enzymatic activity and thus allows greater flexibility with regard to ODN design. The enhanced nucleolytic stability of an ODN with some or all internucleoside linkages modified would be useful for in vivo applications if these compounds are able to form stable triplex structures. The present invention discloses a class of oligonucleotides with enhanced stability for triplex formation.
Candidate oligonucleotides should produce triplex formation at physiologic salt concentrations. The close association of two nucleic acid strands creates a highly negatively charged environment. Many oligonucleotides disclosed in the literature to date require the presence of magnesium ions above physiologic concentrations to produce stable triplex formation. The obligate presence of magnesium ions for triplex association most likely reduces interstrand charge repulsions. However, magnesium concentrations within the cell cannot be altered without altering cell physiology. Therefore, oligonucleotides dependent on minimum concentrations of magnesium ions may not function well in the cellular milieu.
The present invention discloses a class of oligonucleotides that strongly enhance triplex formation even in the presence of K.sup.+ ion concentrations exceeding physiologic levels. This invention promotes oligonucleotide-mediated duplex formation and can be used for antisense technologies.