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 stability and efficient protein synthesis in mRNA-based vaccines and therapeutics, in vitro 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.
How is 5′ capping characterized?
An LC-MS based method, developed by Michael Beverly, et al 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 mass spectrometer.
Where can it be improved?
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.
This method:
- Enables confident identification and sequence confirmation of 5′ digestion products using HRAM MS/MS on Thermo Scientific Orbitrap Exploris 240 mass spectrometer,
- Ensures accurate measurement of capping efficiency
- Provides a streamlined workflow with automated data analysis using Thermo Scientific BioPharma Finder software.
Download the application note to learn more.
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