RNase H-based analysis of synthetic mRNA 5’ cap incorporation

Abstract

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5′ cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5′ capping is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of pre-defined short fragments from the 5′ end of the kilobase-long synthetic mRNA molecules using RNase H or a DNAzyme, 5′ cleavage products enrichment, and LC-MS intact mass analysis. In this communication, we describe 1) our results on the sequence preference of RNase H cleavage and a framework to design site-specific RNA cleavage; 2) a method to fluorescently label the RNase H cleavage fragments for readouts such as easily accessible urea-PAGE or high throughput capillary electrophoresis; 3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5′ cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Biochemical characterization of mRNA capping enzyme from Faustovirus

“…We next verified the presence of the three individual enzyme activities. Using capillary electrophoresis as readout (Chan et al 2022), we subjected FCE to conditions that facilitated the TPase activity, TPase and GTase activities, or all three activities. Using substrate RNA oligonucleotides containing a 5 triphosphate group (ppp-), FCE generated a 5 diphosphate end (pp-) in the absence of GTP and SAM, unmethylated G-capped 5 end (Gppp-) in the presence of GTP only, and Cap-0 N7-methyl G-capped 5 end (m7Gppp-) in the presence of both GTP and SAM (Suppl.…”

“…To assess the RNA capping activity of FCE and VCE on in vitro transcripts, 20 nM FCE or VCE was used to cap 3.5 μM (2 μg/μL) of FLuc transcripts (1.8 kb) containing the 5 sequence of the first 50 nt from human beta globin and synthetic transcripts derived from pRNA21, FLuc, and Comirnaty in 100 μL reactions for 1 hour at 37°C. The extent of cap incorporation was measured using targeted RNase H cleavage and LC-MS (Chan et al 2022). Overall, transcripts containing different 5 sequences were capped to different extent by both enzymes, and FCE generated a higher percentage of m7Gppp-cap than VCE in the in vitro transcripts tested (Figure 10).…”

“…The reactions were carried out in a thermocycler for 30 minutes at a specific temperature and quenched by adding equal volume of a 2x stop solution containing 20 mM EDTA and 2% SDS. The quenched reactions were diluted for capillary electrophoresis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array) using POP-7 polymer and GenScan 120 LIZ dye Size Standard (Chan et al 2022;Wulf et al 2019;Greenough et al 2016). Peak detection and quantification were performed using the Peak Scanner software v.1.0 (Thermo Fisher Scientific) and an in-house data analysis suite.…”

“…Validation of the capillary electrophoresis platform for RNA cap analysis has been published (Chan et al 2022).…”

“…Therefore, 5 cap incorporation is considered one of the critical quality attributes (CQAs) in mRNA manufacturing (Daniel et al 2022). Methods to assess the extent of 5 cap incorporation have been published (Chan et al 2022;Vlatkovic et al 2022;Cairns et al 2003;Beverly et al 2016).…”