Human RNase 4 improves mRNA sequence characterization by LC-MS/MS.

Autor:	Wolf EJ; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Grünberg S; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Dai N; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Chen TH; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Roy B; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Yigit E; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA., Corrêa IR; New England Biolabs, Inc, 43/44 Dunham Ridge, Beverly, MA 01915, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2022 Oct 14; Vol. 50 (18), pp. e106.
DOI:	10.1093/nar/gkac632
Abstrakt:	With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a larger population of uniquely mappable cleavage products. We deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine-two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5' cap incorporation into in vitro transcribed mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS. (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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