Is the contribution of cis and trans protonated 5-methylcytosine-SO3− isomers equal in the conversion to thymine-SO3− under bisulfite conditions? A theoretical perspective

Cytosine (Cyt) can be converted to 5-methylcytosine (5-MeCyt) in CpG sequences of DNA. Conventional bisulfite sequencing can discriminate Cyt from 5-MeCyt, however inappropriate conversion of 5-MeCyt to thymine and a failure to convert Cyt to uracil always occur when Cyt and 5-MeCyt are treated with bisulfite, which would lead to erroneous estimates of DNA methylation densities. Here, the direct hydrolytic deamination of cis (paths A–C) and trans (paths A′–C′) 5-MeCytN3+-SO3− isomers with bisulfite have been explored at the MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p) level. The activation free energies (ΔGs-a≠) of the cis and trans 5-MeCytN3+-SO3− isomers’ paths exhibit no obvious differences, implying both isomers may make an equal contribution to the hydrolytic deamination of 5-MeCyt under bisulfite conditions. It is greatly expected that these results could aid experimental scientists to explore new methods to avoid the formation of the deaminated reactants (5-MeCytN3+-SO3−). Meanwhile, the HSO3−-induced direct hydrolytic deamination of cis and trans 5-MeCytN3+-SO3− isomers is represented by paths A and A′, respectively, and has been further explored in the presence of two water molecules. It was found that the contribution of two water molecules renders the HSO3−-induced direct hydrolytic deamination of cis and trans 5-MeCytN3+-SO3− isomers by paths A and A′ favourable. In addition, the ΔGs-a≠ values (85.74–85.34 kJ mol−1) of the rate-limiting steps of the two water-mediated paths A and A′ are very close to that of the theoretical value for CytN3+-SO3− (88.18 kJ mol−1), implying that the free barrier gap between Cyt and 5-MeCyt is very small under bisulfite conditions. This further suggests that bisulfite sequencing technology may be easily influenced by the external environment.