The deamination mechanism of 5,6‐dihydrocytosine and 5,6‐dihydro‐5‐methylcytosine under typical bisulfite condition

Abstract

AbstractThe discrimination of DNA methylation could make more complicated with the appearance of these minor bases. The conventional bisulfite sequencing faces with the tremendous challenge in the discrimination of DNA methylation. Therefore, the improved and modification bisulfite sequencing has been conducted to raise the reaction selectivity. The key mechanism for these bisulfite sequencing is the reaction of nucleic acids with HSO3− under acidic conditions. The C5═C6 bonds of 5‐methylcytosine (5‐MeCyt) and cytosine (Cyt) have been modified to improve the difference of activation free energies in bisulfite conditions. Because of this, the hydrolytic deamination of DHCytN3+ (paths A and B) and 5‐DHMeCytN3+ isomers (equatorial and axial conformers, paths C and D) has been explored in the presence of the HSO3− group. The activation free energies (ΔGs≠) of DHCytN3+ and 5‐DHMeCytN3+ equatorial conformer paths exhibit no obvious difference, which increases difficulties in differentiating cytosine from 5‐methylcytosine under bisulfite conditions. Meanwhile, the difference of ΔGs≠ between the 5‐DHMeCytN3+ axial conformer and DHCytN3+ is consistent with the experimental results, where the calculated deamination rate is lower for 5,6‐dihydro‐5‐methylcytosine than for 5,6‐dihydrocytosine. And by extrapolation, the differentiating Cyt from 5‐MeCyt may have a greater possibility by bisulfite treatment. These results have high expectations for the experimental scientists to explore new methods to avoid the formation of the 5‐DHMeCytN3+ equatorial conformer. Furthermore, the rate constant with Wigner tunneling correction for paths A, C, and D are in good agreement with uncorrected results.