mRNA Cap Efficiency Detection

The detection of mRNA capping efficiency is a process used to assess whether the cap structure (usually a 7-methylguanosine triphosphate (m7G) cap) on the 5' end of mRNA molecules has been successfully added. This cap structure is crucial for the stability of the mRNA and its translation efficiency within cells. The 5' cap of mRNA plays several key biological functions: regulating nuclear output, preventing nuclease degradation, and promoting translation. The stability in vivo and effective protein synthesis in vitro are essential for ensuring the efficacy of mRNA-based vaccines and therapies. In vitro transcribed (IVT) mRNA needs to be capped at the 5' end through post-transcriptional enzyme capping or co-transcriptional capping. The 5' cap is a critical quality attribute (CQA) of IVT mRNA, which needs to be thoroughly characterized during product and process development.

 

The LC-MS-based method developed by Michael Beverly and others in 2016 has become the standard method for confirming the 5' cap and measuring capping efficiency. In this method, the mRNA is first hybridized with a biotinylated probe complementary to the 5' end of the target mRNA. This probe contains several DNA nucleotides arranged in sequence, which form a DNA:RNA duplex when hybridized with mRNA. When hybridized mRNA undergoes RNase H digestion, the 5' end is cleaved from the mRNA, separated from the digestion mixture using streptavidin-coated magnetic beads, and finally identified and the capping efficiency calculated using high-resolution mass spectrometry.