METHOXYAMINE ATTACK ON CYTOSINE PRODUCES AMBIVALENT BASE-PAIRING PROPERTIES OF THE MODIFIED BASE

We report the solution structure of two heptanucleotides each containi ng a central N-4-methoxycytosine, in one case with adenine on the oppo site strand and in the other with guanine. For the N-4-methoxycytosine -adenine pair only the imino form of the N-4-methoxycytosine residue i s observed and base pairing is in Watson-Crick geometry. However, rota tion of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N-4-met hoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong pre ference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N-4-methoxysytosine base pair ed with guanosine we observe the N-4-methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species wit h an imino form base paired in wobble geometry. The amino form is pred ominant at low temperature whereas the imino form predominates above 4 0 degrees C. Our results point to preferential replacement of dTTP by N-4-methoxycytosine in primer elongation.