Variable presence of 5-methylcytosine in commercial RNA and DNA

Abstract

Nucleoside methylations and other nucleic acid modifications have recently encountered a surge in interest, prompted, among other things, by the detection of methylation and active demethylation of DNA and mRNA by similar mechanisms. In DNA, deoxycytidine methylation by Dnmt enzymes generates 5-methyldeoxycytidine,1 an important epigenetic mark that typically causes inactivation of transcription of the methylated promoter region. Recent exciting developments have shown that these marks are not concrete-cast, but can be actively removed by the oxidative action of TET enzymes,2 which generate, through a series of 2-electron oxidations, first hydroxymethylcytidine (hm5C), then formyldeoxycytidine (f5C),3 and finally carboxydeoxycytidine (ca5C), which may eventually regenerate deoxycytidine by decarboxylation. The apparent functional homolog in mRNA is m6A, which appears to reduce translation efficiency. Here, too, the methylation can be removed by TET-related enzymes generating first hydroxymethyladenosine (hm6A), then formyladenosine (f6A).4,5,6 Also, the presence of 5-methylcytidine in mRNA has been reported early on, and has recently raised renewed interest, although its function is as yet unclear and putative conversion into hydroxymethylcytidine is yet to be demonstrated.32 These developments are enhanced by the development of highly sensitive detection methods7,8 including the adaptation of the so-called bisulfite sequencing from DNA, where it is well established,9 to RNA, where its application has significantly contributed to the present high level of interest.10,11,12 However, bisulfite sequencing alone does not yield unassailable results13,14 and we have thus looked to expand the limits of detection of m5C in both DNA and RNA by LC-MS/MS. Here we report a straightforward regimen that provides values for the limit of quantification (LOQ) in the triple digit attomol range. Its application to presumed negative controls, namely synthetic oligonucleotides, surprisingly detected significant amounts of m5C in both types of synthetic nucleic acids.