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Pharmaceutics
Volume 14
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10.3390/pharmaceutics14020328
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Article

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

by
Irena Vlatkovic
1,*,†,
János Ludwig
2,†,
Gábor Boros
1,
Gábor Tamás Szabó
1,
Julia Reichert
1,
Maximilian Buff
1,
Markus Baiersdörfer
1,
Jonas Reinholz
1,
Azita Josefine Mahiny
1,
Uğur Şahin
1 and
Katalin Karikó
1
1
BioNTech SE, An der Goldgrube 12, 55131 Mainz, Germany
2
Institute of Clinical Chemistry and Clinical Pharmacology, Rheinische Friedrich-Wilhelms-Universität Bonn, 53127 Bonn, Germany
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pharmaceutics 2022, 14(2), 328; https://doi.org/10.3390/pharmaceutics14020328
Submission received: 9 December 2021 / Revised: 24 January 2022 / Accepted: 27 January 2022 / Published: 29 January 2022
(This article belongs to the Special Issue mRNA Therapeutics: A Themed Issue in Honor of Professor Katalin Karikó)
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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.
Keywords: mRNA capping efficiency; ribozyme; in vitro-transcribed (IVT) mRNA; cap; quality control mRNA capping efficiency; ribozyme; in vitro-transcribed (IVT) mRNA; cap; quality control

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MDPI and ACS Style

Vlatkovic, I.; Ludwig, J.; Boros, G.; Szabó, G.T.; Reichert, J.; Buff, M.; Baiersdörfer, M.; Reinholz, J.; Mahiny, A.J.; Şahin, U.; et al. Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics 2022, 14, 328. https://doi.org/10.3390/pharmaceutics14020328

AMA Style

Vlatkovic I, Ludwig J, Boros G, Szabó GT, Reichert J, Buff M, Baiersdörfer M, Reinholz J, Mahiny AJ, Şahin U, et al. Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics. 2022; 14(2):328. https://doi.org/10.3390/pharmaceutics14020328

Chicago/Turabian Style

Vlatkovic, Irena, János Ludwig, Gábor Boros, Gábor Tamás Szabó, Julia Reichert, Maximilian Buff, Markus Baiersdörfer, Jonas Reinholz, Azita Josefine Mahiny, Uğur Şahin, and et al. 2022. "Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA" Pharmaceutics 14, no. 2: 328. https://doi.org/10.3390/pharmaceutics14020328

APA Style

Vlatkovic, I., Ludwig, J., Boros, G., Szabó, G. T., Reichert, J., Buff, M., Baiersdörfer, M., Reinholz, J., Mahiny, A. J., Şahin, U., & Karikó, K. (2022). Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA. Pharmaceutics, 14(2), 328. https://doi.org/10.3390/pharmaceutics14020328

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