Human RNase 4 improves mRNA sequence characterization by LC–MS/MS. Issue 18 (25th July 2022)

• Record Type: Journal Article
• Authors: Wolf, Eric J; Grünberg, Sebastian; Dai, Nan; Chen, Tien-Hao; Roy, Bijoyita; Yigit, Erbay; Corrêa, Ivan R
• Is Part Of: Nucleic acids research. Volume 50:Issue 18(2022)
• Page Start: e106
• Page End: e106
• Publication Date: 2022-07-25
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkac632
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: 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 (m 1 Ψ) or 5-methoxyuridine (mo 5 U), 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.