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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
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.
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.
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