Analogues of the homopyrimidine oiigonucleotide dT,s that contained phosphorothioat¢ bonds of a mixture ofdiastereoisomers or one of tile two stereoisomers (either Rp or Sp) were synthesized. The analogues were mixed under eondition~ conducive to the formation of tripl¢.-.atranded assemblies. The mixtures were characterized by their thermal stabilities (7"1,, values), CD spectra, and gel electropltoresis pattern. The 34-met duplexes containing 15 central purines on one strand attd 15 complementary pyrimidines on the other strand gave no detectable triple helix upon combination with dTt~S~. On the other hand, 34-bp duplexes with dT~sS,, having Rp or Sp, formed triple helix~. This suggests that a sterie factor plays an important role in triple helix formation.
The ability of homopyrimidine oligoribonucleotides (RNA) and oligo-2'-O-methyl-ribonucleotides (2'-O-methyl RNA) containing 8-oxo-adenosine (AOH) and 8-oxo-2'-O-methyl (AmOH) adenosine to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The AOH- and AmOH-substituted RNA and 2'-O-methyl RNA oligonucleotides were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. The substitutions of three cytidine residues with AOH showed higher endonuclease inhibition than the substitution of either one or two cytidine residues with AOH. In particular, the 2'-O-methyl RNA oligonucleotide with only one cytidine substituted with AmOH showed higher endonuclease inhibition than the homopyrimidine RNA and 2'-O-methyl RNA oligonucleotides and the RNA oligonucleotides containing either one or two AOH moieties. Furthermore, the AmOH-substituted 2'-O-methyl RNA oligonucleotides were stable (53%) after an incubation in 10% fetal bovine serum for 8 h, whereas the RNA oligonucleotides were completely degraded. Increased resistance to nucleases is observed with the introduction of 2'-O-methylnucleosides. This stabilization should help us to design much more efficient third strand homopyrimidine oligomer and antisense nucleic acid-based antiviral therapies, which could be used as tools in cellular biology.
The ability of homopyrimidine oligonucleotides containing 8-oxo-2'-deoxyadenosine to form stable, triple helical structures with the sequence containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-0x0-2'-deoxyadenosinesubstituted oligomers were shown to inhibit enzymatic cleavage and to bind within the physiological pH range in a pa-independent fashion without compromising specificity.
Reverse transcription of HIV-1 vRNA into the double-stranded DNA provirus involves initiation of plus-strand DNA synthesis at the polypurine tract (PPT) by reverse transcriptase (RT). The PPT is highly conserved among the known human immunodeficiency virus (HIV-1) strains and is a possible target for triplex formation. We show the effects of triple-helix formation by assays of primer extension inhibition in vitro, using a two-strand system (foldback triplex-forming oligonucleotides (FTFOs)) targeted to the PPT of HIV-1. The two-stranded composition of a triple-helix is thermodynamically and kinetically superior to the three-strand system. The FTFOs inhibited the RT activity in a sequence-specific manner, i.e. the triplex actually formed at the PPT and blocked the RT. The FTFOs containing the phosphorothioate groups at the antisense sequences showed greater 3P P-exonuclease resistance. In HIV-1-infected MOLT-4 cells, the FTFOs containing the phosphorothioate groups at the antisense sequence sites and guanosine rich parts within the third Hoogsteen base-pairing sequence inhibit the replication of HIV-1 more effectively than the antisense oligonucleotides, indicating sequence-specific inhibition of HIV-1 replication.z 1999 Federation of European Biochemical Societies.
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