Dataset Information

Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

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.

SUBMITTER: Nwokeoji AO

PROVIDER: S-EPMC9872168 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

Nwokeoji Alison Obinna AO Chou Tachung T Nwokeoji Eleojo Ahuva EA

ACS synthetic biology 20221210 1

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 deve ...[more]

PMID: 36495278

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Project description:Background. The gram-negative Neisseria is associated with two of the most potent human epidemic diseases: meningococcal meningitis and gonorrhoea. In both cases, disease is caused by bacteria colonizing human mucosal membrane surfaces. Overall, the genus shows great diversity and genetic variation mainly due to its ability to acquire and incorporate genetic material from a diverse range of sources through horizontal gene transfer. Although a number of databases exist for the Neisseria genomes, they are mostly focused on the pathogenic species. In this present study we present the freely available NeisseriaBase, a database dedicated to the genus Neisseria encompassing the complete and draft genomes of 15 pathogenic and commensal Neisseria species. Methods. The genomic data were retrieved from National Center for Biotechnology Information (NCBI) and annotated using the RAST server which were then stored into the MySQL database. The protein-coding genes were further analyzed to obtain information such as calculation of GC content (%), predicted hydrophobicity and molecular weight (Da) using in-house Perl scripts. The web application was developed following the secure four-tier web application architecture: (1) client workstation, (2) web server, (3) application server, and (4) database server. The web interface was constructed using PHP, JavaScript, jQuery, AJAX and CSS, utilizing the model-view-controller (MVC) framework. The in-house developed bioinformatics tools implemented in NeisseraBase were developed using Python, Perl, BioPerl and R languages. Results. Currently, NeisseriaBase houses 603,500 Coding Sequences (CDSs), 16,071 RNAs and 13,119 tRNA genes from 227 Neisseria genomes. The database is equipped with interactive web interfaces. Incorporation of the JBrowse genome browser in the database enables fast and smooth browsing of Neisseria genomes. NeisseriaBase includes the standard BLAST program to facilitate homology searching, and for Virulence Factor Database (VFDB) specific homology searches, the VFDB BLAST is also incorporated into the database. In addition, NeisseriaBase is equipped with in-house designed tools such as the Pairwise Genome Comparison tool (PGC) for comparative genomic analysis and the Pathogenomics Profiling Tool (PathoProT) for the comparative pathogenomics analysis of Neisseria strains. Discussion. This user-friendly database not only provides access to a host of genomic resources on Neisseria but also enables high-quality comparative genome analysis, which is crucial for the expanding scientific community interested in Neisseria research. This database is freely available at http://neisseria.um.edu.my.

Dataset Information

Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

Publications

Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

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