Inhibition of Restriction Endonuclease Cleavage by Triple Helix Formation with RNA and 2‘-O-Methyl RNA Oligonucleotides Containing 8-Oxo-adenosine in Place of Cytidine

Abstract

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.

Post‐transcriptional regulation of gene expression by degradation of messenger RNAs

“…2 0 -O-methyl oligoribonucleotides (ORNs) are gaining widespread attention because of their newly discovered, diverse biomedical applications in viral and cancer therapies (Wan et al, 1998;Ushijima et al, 1999). RNA hybridized to 2 0 -O-methyl ORNs and incubated in nuclear extracts from HeLa cells was truncated by the inhibition of an exonuclease degrading RNA in the 3 0 to 5 0 direction (Dominski et al, 1996).…”

Transplatin-Conjugated Triplex-Forming Oligonucleotides Form Adducts with Both Strands of DNA

“…The TFOs that consisted of deoxyribonucleotides, 1d , 2d , and 3d , formed relatively stable triplexes whose third strands dissociated at approximately 50°C, a temperature lower than the melting temperature of duplex 4RY , 65°C. We and others have shown previously that incorporation of 2′- O -methylribonucleotides can stabilize TFO binding (40-45). In accord with these findings, the TFOs containing 2′- O -methylribonucleotides, 2mr and 3mr , formed triplexes that melted with a single transition at greater than 70°C.…”

8‐Oxo‐7,8‐dihydroadenine and 8‐Oxo‐7,8‐dihydroadenosine—Chemistry, Structure, and Function in RNA and Their Presence in Natural Products and Potential Drug Derivatives

“…[52] The properties of the novel oligonucleotides were tested in the inhibition of the sequence specific cleavage of simianv irus 40 DNA by endonuclease Ksp632-I. [52] The properties of the novel oligonucleotides were tested in the inhibition of the sequence specific cleavage of simianv irus 40 DNA by endonuclease Ksp632-I.…”

“…Ushijima et al then reported on the incorporation of 8-oxoA into oligonucleotides of RNA (using the canonical 3'-5'-linkage). [52] The properties of the novel oligonucleotides were tested in the inhibition of the sequence specific cleavage of simianv irus 40 DNA by endonuclease Ksp632-I. Between 1-3 modifications were incorporated to observe decreased T m valuesw ith increasing number of 8-oxoA units present within the formed triple-helix structures.P ossibilities for base pairing interactions with G, C, and Twere proposed as aconformational change between anti!syn is possible, with the syn conformationb eing the preferred form.…”