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Related Experiment Video

Updated: May 28, 2025

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Published on: December 9, 2022

Mass spectrometry-based mRNA sequence mapping via complementary RNase digests and bespoke visualisation tools.

Emma N Welbourne¹, Royce J Copley¹, Gareth R Owen¹

¹School of Chemical, Materials and Biological Engineering, University of Sheffield, Sheffield, UK. m.dickman@sheffield.ac.uk.

|February 10, 2025

Summary

This summary is machine-generated.

This study introduces a new method using partial RNA digests and novel software for rapid mRNA sequencing. This approach significantly improves sequence coverage and analysis accuracy for mRNA therapeutics.

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Area of Science:

Biochemistry
Molecular Biology
Bioinformatics

Background:

Messenger RNA (mRNA) technology has revolutionized vaccine development, reducing timelines significantly.
Accurate characterization of mRNA therapeutics is crucial for manufacturing and quality control.

Purpose of the Study:

To develop and validate a rapid, high-throughput workflow for characterizing mRNA therapeutics.
To create novel software for optimizing the analysis and visualization of mRNA sequence mapping.

Main Methods:

Utilized partial RNase T1 and RNase U2 digests of mRNA.
Implemented an automated, high-throughput workflow for analysis.
Developed custom software for LC-MS/MS data processing and visualization.

Main Results:

Achieved combined mRNA sequence coverage of 64% with multiple RNase T1 digests and 88% with combined RNase T1 and U2 digests.
The developed software automates digest combination, enhancing analysis speed and accuracy.
Novel visualization tools, including spiral plots, improve sequence map analysis.

Conclusions:

The integrated workflow and software enable rapid and accurate sequence mapping of mRNA therapeutics.
This method enhances confidence in sequence mapping through increased coverage and overlapping fragment analysis.
The approach facilitates efficient characterization critical for mRNA therapeutic development.