Effect of tautomeric shift on mutation: N4-methoxycytidine forms hydrogen bonds with adenosine in polymers.

N4-Methoxycytidine (mo4C), previously found to act only as uridine (U) in transcription [Singer, B., & Spengler, S. (1981) Biochemistry 20, 1127], was tested for its ability to base pair as U in copolymers of (U,mo4C) annealed with poly(A) or transcribed with ATP and DNA-dependent RNA polymerase. Mixing curves have now indicated that the derivative is retained in a poly(U,39% mo4C).poly(A) helix, unlike unmodified C in poly(U,35% C). The presence of 13-39% mo4C in U polymers lowered the melting temperature, Tm, observed in annealed complexes both with poly(A) and after transcription with ATP. However, complexes isolated after transcription had a large hyperchromicity and melted cooperatively, which indicated that they are hydrogen bonded. The decreased Tm for poly(U,mo4C).poly(A) compared to that for poly(U).poly(A) can be attributed to stacking changes and adjacent base-pair disruption by mo4C. The greater cooperative melting of transcribed poly(U,39% mo4C) as compared to the annealed complex may indicate that the methoxy substituent is normally a mixture of rotamers and that the syn rotamer is required for transcription. The interference of the methoxy substituent was also shown by the loss of helix formation by poly(C,mo4C) in acid solution. mo4C decreased the Tm much more than A, which stacks well in acid. U, which neither stacks nor participates in an acid structure, caused more distortion than either of the other bases. It is inferred that mo4C has the base-pairing ability of U but that the planarity of the substituent is lost.