METHOXYAMINE-INDUCED MUTAGENESIS OF NUCLEIC-ACIDS - A PROTON NMR-STUDY OF OLIGONUCLEOTIDES CONTAINING N-4-METHOXYCYTOSINE PAIRED WITH ADENINE OR GUANINE

We report the solution structure of two heptanucleotides each containi ng a central N-4-methoxycytosine, in one case paired with adenine on t he opposite strand and the other with guanine. For the N-4-methoxycyto sine adenine pair, only the imino form of the N-4-methoxycytosine resi due is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constraine d to a particular orientation although it must be anti to the N3 of N- 4-methoxycytosine. The slow exchange on a proton NMR time scale betwee n the single strand and double strand forms is attributed to the stron g preference of the cis conformation of the OCH, group in the single s trand, which inhibits base pair formation. For the N-4-methoxycytosine that is base paired with guanine, we observe an amino form in Watson- Crick geometry in slow exchange with a base paired imino form in wobbl e geometry. The amino form is predominant at low temperature whereas t he imino form predominates above 313 K. We have measured the exchange rate between the two forms at 303 K and observed a value of approximat ely 1 s(-1). The relative ratio of amino and imino forms of N-4-methox ycytosine is influenced by both the base that is in front and the temp erature. Our results explain the preferential replacement of dTTP by N -4-methoxycytosine in primer elongation.