Variable presence of 5-methylcytosine in commercial RNA and DNA

5-methylcytosine (m⁵C, mC) is a naturally occurring nucleoside modification in both RNA and DNA. Its presence in DNA is a widely accepted epigenetic mark for transcription inactivation. In RNA, its appearance in different coding as well as non-coding RNA implies multiple functions, with regulation of gene expression as a common denominator. Here we report on the serendipitous discovery of m⁵C in synthetic oligonucleotides, which prompted a systematic quantification in synthetic DNA and RNA of academic as well as of commercial origin. For both types of oligonucleotides, m⁵C was identified by comparison of fragmentation pattern and retention time with authentic standards by highly sensitive LC-MS/MS analysis with limit-of-quantification values in the attomol range. Quantification revealed m⁵C contents ranging from the ppm range to single digit percentiles for certain commercial suppliers, while others remained below the single digit promille mark. The only oligonucleotides without m⁵C were entirely devoid of cytosines in their sequence, thus tying m⁵C occurance to the use of cytosine phosphoramidites during synthesis. These findings may have important bearings for the use of synthetic oligonucleotides under such diverse aspects as gene expression, immunostimulation, or biophysical studies of duplex stability.