{"id":5007,"date":"2022-08-10T00:00:00","date_gmt":"2022-08-10T00:00:00","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/simplifying-mrna-5-cap-characterization\/"},"modified":"2026-09-09T01:03:56","modified_gmt":"2026-09-09T01:03:56","slug":"simplifying-mrna-5-cap-characterization","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/simplifying-mrna-5-cap-characterization\/","title":{"rendered":"Simplifying mRNA 5′ Cap Characterization"},"content":{"rendered":"
\n

Why is the 5′ capping important? The 5′ cap of mRNA plays multiple key biological functions: regulation of nuclear export, prevention of degradation by exonucleases, and promotion of translation. To ensure in vivo<\/em> stability and efficient protein synthesis in mRNA-based vaccines and therapeutics, in vitro<\/em> transcribed (IVT) mRNAs need to be capped at the 5′ end through either post-transcription enzymatic capping or co-transcriptional capping. The 5′ cap is a critical quality attribute (CQA) for IVT mRNAs and needs to be fully characterized during product and process development. A robust assay is required to confirm the identity of 5′ cap and accurately measure the capping efficiency.<\/p>\n

How is 5′ capping characterized?<\/strong><\/h3>\n

An LC-MS based method, developed by Michael Beverly, et al<\/a> in 2016, has become the standard assay to confirm the identity of the 5′ cap and measure the capping efficiency. In this method, mRNAs were first complexed with a biotinylated probe complementary to the 5′ end of the target mRNA. The probe contained several DNA nucleotides in sequence, that when hybridized to the mRNA, created a DNA:RNA duplex. When the complexed mRNA was subjected to an RNase H digestion, the 5′ end was cleaved from the mRNA, isolated from the digestion mixture using streptavidin-coated magnetic beads, and identified using high-resolution accurate-mass MS-only scans on a Thermo Scientific Q Exactive <\/a>mass spectrometer.<\/p>\n

\"\" <\/span>Where can it be improved?<\/strong><\/h3>\n

The incorporation of sequence mapping and automated data processing can dramatically improve identification confidence and overall throughput. Robert Ross, senior product applications specialist at Thermo Fisher Scientific, recently developed a sensitive and robust LC-HRAM MS\/MS method for identification, sequence confirmation, and relative quantitation of the 5′ cap in IVT mRNA products.<\/p>\n

This method:<\/p>\n