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Related Concept Videos

RNA-seq03:21

RNA-seq

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Related Experiment Video

Updated: Sep 3, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
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Human RNase 4 improves mRNA sequence characterization by LC-MS/MS.

Eric J Wolf1, Sebastian Grünberg1, Nan Dai1

  • 1New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA.

Nucleic Acids Research
|July 24, 2022
PubMed
Summary

Human endoribonuclease hRNase 4 enhances messenger RNA (mRNA) analysis. This enzyme improves sequence coverage and modification assessment for mRNA therapeutics and vaccines using liquid chromatography-mass spectrometry.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Synthetic messenger RNA (mRNA) therapeutics and vaccines are rapidly advancing.
  • Accurate characterization of long, complex mRNA molecules is crucial for their development.
  • Existing analytical tools require RNA conversion or amplification, limiting direct assessment.

Purpose of the Study:

  • To evaluate the utility of tandem liquid chromatography-mass spectrometry (LC-MS/MS) for direct mRNA analysis.
  • To investigate the performance of the uridine-specific endoribonuclease hRNase 4 in mRNA characterization.
  • To assess hRNase 4's capability in analyzing modified mRNAs and 5' cap incorporation.

Main Methods:

  • Digestion of mRNA using site-specific endoribonucleases to produce short oligonucleotides.
  • Analysis of oligonucleotide pools using tandem liquid chromatography-mass spectrometry (LC-MS/MS).
  • Comparison of hRNase 4 with the benchmark enzyme RNase T1 for mRNA sequence coverage.

Main Results:

  • hRNase 4 generated a larger population of uniquely mappable cleavage products compared to RNase T1, improving mRNA sequence coverage.
  • hRNase 4 successfully characterized mRNAs with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U) substitutions.
  • hRNase 4 enabled direct assessment of 5' cap incorporation in in vitro transcribed mRNA.

Conclusions:

  • hRNase 4 is a powerful tool for interrogating mRNA sequence, identity, and modifications via LC-MS/MS.
  • The enzyme enhances the analysis of modified mRNAs, crucial for reducing immunogenicity.
  • hRNase 4 facilitates direct assessment of mRNA integrity, including 5' cap status.