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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...

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Autores principales: Vlatkovic, Irena, Ludwig, János, Boros, Gábor, Szabó, Gábor Tamás, Reichert, Julia, Buff, Maximilian, Baiersdörfer, Markus, Reinholz, Jonas, Mahiny, Azita Josefine, Şahin, Uğur, Karikó, Katalin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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.
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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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