Methoxyamine attack on cytosine produces ambivalent base pairing properties of the modified base

We report the solution structure of two heptanucleotides each containing a central N⁴-methoxycytosine, in one case with adenine on the opposite strand and in the other with guanine. For the N⁴-methoxycytosine-adenine pair only the imino form of the N⁴-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation 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 N⁴-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the syn conformation of the OCH3 group in the single strand which inhibits base pair formation. For N⁴-methoxycytosine base paired with guanosine we observe the N⁴-methoxycytosine base in the amino form in Watson-Crick geometry and a slow exchange of this species with an imino form base paired in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 40°C. Our results point to preferential replacement of dTTP by N⁴-methoxycytosine in primer elongation.