Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

Abstract

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.

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

“…It has been reported recently that ribozyme-cleaved 5’ cleavage fragments of in vitro transcript could be enriched for LC-MS analysis using silica-based spin columns. The spin column format, however, requires 20 – 60 μg RNA input (Vlatkovic et al 2022). The use of RNase H and biotinylated targeting oligos allows for affinity enrichment (Beverly et al 2016) that could potentially decrease the input RNA requirement.…”

“…Analytical methods such as gel-electrophoresis and mass spectrometry may lack the resolution or detection range to confidently distinguish between Cap-1 or Cap-0 capped RNA and unreacted or intermediate capping products on full-length synthetic mRNA molecules. To overcome this problem, methods that cleave a pre-defined fragment from the 5’ end of the mRNA molecule using custom designed DNAzyme (Cairns et al 2003), ribozyme (Vlatkovic et al 2022) or DNA-RNA chimera-guided RNase H (Lapham and Crothers 1996; Yu et al 1997; Lapham et al 1997) have been reported. The 5’ cleavage fragments, ranging from 5 to 30 nucleotides long, can be readily analyzed by denaturing gel electrophoresis or liquid chromatography-mass spectrometry (LC-MS) (Beverly et al 2016).…”

“…However, there has been insufficient guidance on the DNA-RNA chimera design to achieve uniform RNase H cleavage, and the readout method is limited to resource-intensive LC-MS. Recently, Vlatkovic et al showed that custom designed ribozymes can generate uniform 5’ fragments for cap analysis and that the 5’ cleavage fragments can be purified by silica-based spin columns (Vlatkovic et al 2022). In this communication, by applying a DNA-RNA chimera selection framework that screens for RNase H specificity empirically, we showed that RNase H can generate highly uniform cleavage at pre-defined sites.…”

“…Because the fill-in activity is dependent on the complementarity of the incoming fluorescently labeled dNTPs to the DNA-RNA chimera, the labeling step further constraints the size of the fluorescently labeled RNase H cleavage product to effectively eliminate non-target cleavage products from mobility-based methods such as gel- or capillary electrophoresis. Together with the DNAzyme- and ribozyme-based methods (Cairns et al 2003; Vlatkovic et al 2022), we hope that our methods (Figure 2) can help make RNA cap analysis more accessible to the scientific community.…”

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

“…Analytical methods such as gel electrophoresis and mass spectrometry may lack the resolution or detection range to confidently distinguish between Cap-1 or Cap-0 capped RNA and unreacted or intermediate capping products on full-length synthetic mRNA molecules. To overcome this problem, methods that cleave a predefined fragment from the 5′ end of the mRNA molecule using custom designed DNAzyme ( Cairns et al 2003 ), ribozyme ( Vlatkovic et al 2022 ), or DNA–RNA chimera-guided RNase H ( Lapham and Crothers 1996 ; Lapham et al 1997 ; Yu et al 1997 ) have been reported. The 5′ cleavage fragments, ranging from 5 to 30 nt long, can be readily analyzed by denaturing gel electrophoresis or liquid chromatography-mass spectrometry (LC-MS) ( Beverly et al 2016 ).…”