Jin Lingxia, Wang Lu, Zhang Caiying, Wang Wenliang, Min Suotian, Hu Daodao
Key Laboratory for Macromolecular Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
Phys Chem Chem Phys. 2014 Aug 14;16(30):16264-77. doi: 10.1039/c4cp00387j.
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.
胞嘧啶（Cyt）可在DNA的CpG序列中转化为5-甲基胞嘧啶（5-MeCyt）。传统的亚硫酸氢盐测序能够区分Cyt和5-MeCyt，然而，当用亚硫酸氢盐处理Cyt和5-MeCyt时，总是会出现5-MeCyt不适当转化为胸腺嘧啶以及Cyt未能转化为尿嘧啶的情况，这会导致对DNA甲基化密度的错误估计。在此，在MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p)水平上研究了顺式（路径A-C）和反式（路径A'-C'）5-MeCytN3(+)-SO3(-)异构体与亚硫酸氢盐的直接水解脱氨反应。顺式和反式5-MeCytN3(+)-SO3(-)异构体路径的活化自由能（ΔG(s-a≠)）没有明显差异，这意味着在亚硫酸氢盐条件下，两种异构体可能对5-MeCyt的水解脱氨反应做出同等贡献。非常期望这些结果能够帮助实验科学家探索新方法，以避免形成脱氨反应物（5-MeCytN3(+)-SO3(-)）。同时，亚硫酸氢根离子（HSO3(-)）诱导的顺式和反式5-MeCytN3(+)-SO3(-)异构体的直接水解脱氨反应分别由路径A和A'表示，并且在存在两个水分子的情况下进行了进一步研究。结果发现，两个水分子的作用使得通过路径A和A'的HSO3(-)诱导的顺式和反式5-MeCytN3(+)-SO3(-)异构体的直接水解脱氨反应变得有利。此外，两条水介导路径A和A'的限速步骤的ΔG(s-a≠)值（85.74 - 85.34 kJ mol(-1)）与CytN3(+)-SO3(-)的理论值（88.18 kJ mol(-1)）非常接近，这意味着在亚硫酸氢盐条件下，Cyt和5-MeCyt之间的自由能垒差距非常小。这进一步表明亚硫酸氢盐测序技术可能很容易受到外部环境的影响。
