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Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA
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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8879150/ https://www.ncbi.nlm.nih.gov/pubmed/35214060 http://dx.doi.org/10.3390/pharmaceutics14020328 |
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author | Vlatkovic, Irena Ludwig, János Boros, Gábor Szabó, Gábor Tamás Reichert, Julia Buff, Maximilian Baiersdörfer, Markus Reinholz, Jonas Mahiny, Azita Josefine Şahin, Uğur Karikó, Katalin |
author_facet | Vlatkovic, Irena Ludwig, János Boros, Gábor Szabó, Gábor Tamás Reichert, Julia Buff, Maximilian Baiersdörfer, Markus Reinholz, Jonas Mahiny, Azita Josefine Şahin, Uğur Karikó, Katalin |
author_sort | Vlatkovic, Irena |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8879150 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-88791502022-02-26 Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA Vlatkovic, Irena Ludwig, János Boros, Gábor Szabó, Gábor Tamás Reichert, Julia Buff, Maximilian Baiersdörfer, Markus Reinholz, Jonas Mahiny, Azita Josefine Şahin, Uğur Karikó, Katalin Pharmaceutics Article 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. MDPI 2022-01-29 /pmc/articles/PMC8879150/ /pubmed/35214060 http://dx.doi.org/10.3390/pharmaceutics14020328 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Vlatkovic, Irena Ludwig, János Boros, Gábor Szabó, Gábor Tamás Reichert, Julia Buff, Maximilian Baiersdörfer, Markus Reinholz, Jonas Mahiny, Azita Josefine Şahin, Uğur Karikó, Katalin Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title | Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title_full | Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title_fullStr | Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title_full_unstemmed | Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title_short | Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA |
title_sort | ribozyme assays to quantify the capping efficiency of in vitro-transcribed mrna |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8879150/ https://www.ncbi.nlm.nih.gov/pubmed/35214060 http://dx.doi.org/10.3390/pharmaceutics14020328 |
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