Evaluation of mRNA Capping Efficiency by Liquid Chromatography Coupled to High Resolution and Accurate Mass Spectrometry

5' capping structure for a massager RNA (mRNA) is critical for its translation level. An “antireverse” cap analog (ARCA, 3´-O-Me-m7G(5')ppp(5')G) has been wildly used to ensure that cap at 5’ terminal is incorporated in the correct orientation during transcription from a DNA template. However, the mRNA produced in this way is not 100% capped due to the competition between guanosine triphosphate (GTP) and ARCA. The liquid chromatography coupled to high resolution and accurate mass spectrometry (LC-HRAM) could be used to differentiate and quantitate the uncapped and capped mRNAs from their 5’ molecules. Here, a method was described to evaluate the capping efficiency of a 36 nucleotides (nt) mRNA by LC-HRAM, which provided the required resolution and accuracy.