Analytical methods for estimation of the mRNA capping efficiency - A comprehensive review.

Capping efficiency is a critical quality attribute of mRNA therapeutics, however its accurate assessment remains a nontrivial analytical challenge. The primary difficulty arises from the minimal contribution of cap structure to the overall molecular mass, since for mRNAs spanning several hundred to thousands of nucleotides. As a result, distinguishing between capped and uncapped species requires multistep sample preparation and highly sensitive analytical instrumentation. Additional complexity is introduced by heterogeneity at the 5' end, which may arise during transcription, capping, and subsequent analytical procedures. In this work, we provide a comprehensive overview of current analytical strategies for the quantitative assessment of mRNA capping efficiency and cap composition characterization. We also discuss sources of 5'-end heterogeneity identified and characterized in methodological studies. This review will enable researchers to identify the most effective strategy for evaluating RNA capping efficiency based on their objectives and the phase of mRNA therapeutic development.