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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Updated: Mar 24, 2026

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Published on: July 10, 2020

Automated Online Direct mRNA Sequence Mapping Using Partial RNase T1 Digests.

Jessica S Dale¹, Emma N Welbourne¹, Caroline A Evans¹

¹School of Chemical, Materials and Biological Engineering, University of Sheffield, Sheffield S1 3JD, U.K.

Analytical Chemistry

|March 23, 2026

Summary

This summary is machine-generated.

Mass spectrometry enables direct mRNA sequence mapping and quality attribute analysis. This automated method provides high sequence coverage rapidly, improving mRNA medicine quality control.

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Area of Science:

Biochemistry
Analytical Chemistry
Molecular Biology

Background:

Mass spectrometry is crucial for analyzing mRNA medicine critical quality attributes (CQAs).
CQAs like sequence identity, 5' capping, and 3' poly(A) tail length significantly affect mRNA medicine quality, safety, and efficacy.
Existing methods for mRNA analysis can be complex and time-consuming.

Purpose of the Study:

To develop and validate a streamlined, automated method for direct mRNA sequence mapping and multi-attribute monitoring.
To enhance the analysis of critical quality attributes for mRNA medicines using mass spectrometry.

Main Methods:

Utilized online partial RNase T1 digests coupled with two-dimensional liquid chromatography-mass spectrometry (2D LC-MS).
Employed automated, on-column RNase digestion with ion-pair reversed-phase liquid chromatography, eliminating manual sample manipulation.
Applied high-resolution tandem mass spectrometry for oligoribonucleotide identification and mRNA sequence mapping.

Main Results:

Achieved high sequence coverage (93-99%) for eGFP mRNA in under 60 minutes.
Demonstrated a fully automated workflow for direct mRNA sequence mapping with high throughput.
Successfully monitored multiple attributes including 5' capping efficiency and 3' poly(A) tail length and heterogeneity within the same workflow.

Conclusions:

The online partial RNase T1 digest coupled with 2D LC-MS offers a rapid, automated, and reproducible method for direct mRNA sequence mapping.
This approach presents significant advantages over existing methods for automated mRNA identity testing and multi-attribute analysis.
Precise control over digest conditions allows for comprehensive quality assessment of mRNA medicines.