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 (ΔG(s-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 ΔG(s-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.