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.
Highlights
In vitro-transcribed (IVT) mRNA-based therapeutics are emerging as novel biologics with a variety of applications, including recent vaccines against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and other infectious disease vaccine candidates, cancer immunotherapies, gene-editing treatments, and protein replacement therapies [1,2,3,4,5,6]
Templates for in vitro transcription were generated by linearizing plasmids containing different coding sequences, flanked by sequences corresponding to the 50 untranslated region (UTR) of human alpha globin or 50 -leader of tobacco etch virus (TEV), a constant 30 UTR, and 100 nt long poly(A) tail [15,16]
Rz5 contained inosine (I) which allows for recognition and cleavage after the ACA triplet in the TEV 50 untranslated regions (50 UTRs) [21]
Summary
In vitro-transcribed (IVT) mRNA-based therapeutics are emerging as novel biologics with a variety of applications, including recent vaccines against coronavirus disease 2019. (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and other infectious disease vaccine candidates, cancer immunotherapies, gene-editing treatments, and protein replacement therapies [1,2,3,4,5,6]. The presence of the cap structure at its 5’ terminus is one of the key features of IVT mRNA that increases its stability and translatability. Capped mRNAs are generally translated more efficiently compared to uncapped mRNAs [7,8,9]. Enzymatic and co-transcriptional capping of IVT mRNA may be incomplete, leading to the presence of a variable number of uncapped molecules in the final IVT mRNA. We developed assays that allow for fast and simple quantitative measurements of the capping efficiency
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