Collection of Czechoslovak Chemical Communications - digital archive 

Abstract

The availability of nucleoside analogues with ambiguous base-pairing properties would be of considerable value in molecular biology. We have incorporated deoxynebularine [9-(2-deoxy-β-D-ribofuranosyl)purine, P], deoxyinosine [9-(2-deoxy-β-D-ribofuranosyl)-6-hydroxypurine, I) and [9-(2-deoxy-β-D-ribofuranosyl)-6-methoxyaminopurine, ^MeOA] into hexadecamer oligodeoxyribonucleotides and tested their behaviour in DNA•DNA hybridisations in vitro, as well as in oligonucleotide-directed mutagenesis experiments in vivo. The results showed that P behaved as an adenine analogue in all assays. Oligonucleotide duplexes containing I/C or I/T base pairs displayed similar thermal stabilities in DNA•DNA hybridisation experiments, however, during DNA synthesis in vitro and in vivo, hypoxanthine behaved strictly as a guanine analogue. Only ^MeOA was truly ambiguous in all assays. The ¹H NMR spectrum of the nucleoside demonstrated the existence of two distinct tautomeric forms in a ratio of ca 8 : 2, implying that the base might pair with both C and T. Indeed, within the context of synthetic hexadecamer duplexes, ^MeOA/C and ^MeOA/T pairs brought about a similar thermal destabilisation, with the former base pair being only marginally less favoured. When used as hybridisation probes on single-stranded M13 DNA, the ^MeOA-containing hexadecamer oligonucleotides were shown to bind with similar efficiencies to target sequences containing either C or T opposite the analogue. Interestingly, when ^MeOA is in the template strand during DNA replication, the polymerase III holoenzyme of E. coli reads it predominantly as a G, which indicates that ^MeOA exists in B-DNA mostly as the anti-imino tautomer.

Keywords: Nucleoside analogues; Ambiguous base-pairing; N⁶-Methoxyadenine; Tautomerism; E. coli mismatch repair system; Nucleosides; Nucleotides; DNA.

References: 27 live references.