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Updated: May 29, 2026
05:41
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
Transcript-independent quantitative analysis of RNA cap structures by LC-MS/MS.
Ryan Kilburn1, Rachel C Gomez1, Steven Evans1
1Department of Research and Development, Nature's Toolbox Inc, Rio Rancho, NM, USA.
Molecular Therapy. Advances
|May 28, 2026
Summary
A new LC-MS/MS assay accurately quantifies RNA 5' cap incorporation, a crucial quality attribute for RNA therapeutics. This transcript-independent method supports process development and quality control for improved RNA drug manufacturing.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Accurate assessment of 5' cap incorporation is vital for RNA therapeutic quality.
- Current methods are often transcript-specific, limiting throughput and broad applicability.
Purpose of the Study:
- To develop a quantitative, transcript-independent LC-MS/MS assay for RNA cap incorporation.
- To support RNA therapeutic process development and quality control.
Main Methods:
- Utilized nuclease P1 digestion and external calibration with cap analog standards.
- Employed paired digested and undigested analyses for reagent correction.
- LC-MS/MS assay with a 10-minute acquisition time.
Main Results:
- Demonstrated excellent linearity (r² > 0.995), accuracy (84%-102% recovery), and precision (RSD <15%).
- Assay supports a 0.01-20 pmol calibration range.
- Validated for multiple RNA transcripts, showing sequence-agnostic quantification.
Conclusions:
- Developed a rigorously characterized, sequence-agnostic analytical platform for RNA cap analysis.
- Method is compatible with chemical and enzymatic capping strategies.
- Enables scalable process development, lot comparability, and manufacturing quality control for RNA therapeutics.
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