Nwokeoji, A.O. orcid.org/0000-0002-7257-8085, Chou, T. and Nwokeoji, E.A. (2023) Low resource integrated platform for production and analysis of capped mRNA. ACS Synthetic Biology, 12 (1). pp. 329-339. ISSN 2161-5063
Abstract
The existing platform for large-scale mRNA production is fast, but consumable costs, process technicality, and complexity represent key bottlenecks limiting global mRNA biologics manufacturing. Another challenge is the lack of a consolidated platform for mRNA product characterization and assays that meet regulatory requirements. Bridging these innovation gaps to simplify processes and reduce cost would improve mRNA biologics manufacturability, especially in low-resource settings. This study develops a "cotranscriptional"capping strategy that utilizes T7 RNA polymerase, and the Vaccinia Capping System to synthesize and cap mRNA. We created an "integrated reaction buffer"that supports both capping enzymes for catalytic and in vitro transcription processes, enabling one-pot, two-step capped mRNA synthesis. Additionally, we report a novel, one-step analytic platform for rapid, quantitative, capped mRNA analysis. The assay involves target mRNA segment protection with cheap DNA primers and RNase digest of non-hybridized or non-target sequences before analysis by single nucleotide-resolving urea-polyacrylamide gel electrophoresis (PAGE). The integrated approach simplifies production processes and saves costs. Moreover, this assay has potential applications for mRNA analyses and post-transcriptional modification detection in biological samples. Finally, we propose a strategy that may enable unparalleled sequence coverage in RNase mass mapping by adapting the developed assay and replacing urea-PAGE with mass spectrometry.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 The Authors. Published by American Chemical Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | capped mRNA; mRNA biologics; integrated biomanufacturing platform; in vitro transcription; Vaccinia Capping System |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 30 Jan 2023 11:36 |
Last Modified: | 30 Jan 2023 11:36 |
Published Version: | http://dx.doi.org/10.1021/acssynbio.2c00609 |
Status: | Published |
Publisher: | American Chemical Society (ACS) |
Refereed: | Yes |
Identification Number: | 10.1021/acssynbio.2c00609 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:195774 |