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. 2022 May 24;94(20):7339-7349.
doi: 10.1021/acs.analchem.2c00765. Epub 2022 May 12.

Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry

Christina J Vanhinsbergh  1 , Angela Criscuolo  2 , Jennifer N Sutton  3 , Keeley Murphy  3 , Andrew J K Williamson  4 , Ken Cook  4 , Mark J Dickman  1
Affiliations

Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry

Christina J Vanhinsbergh et al. Anal Chem. .

Abstract

Large RNA including mRNA (mRNA) has emerged as an important new class of therapeutics. Recently, this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study, we have developed an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilized on magnetic particles were performed in conjunction with high-resolution liquid chromatography-mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach, a >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS-CoV-2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation, can be completed within 90 min. The ability to rapidly identify, characterize, and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation, and impurity analysis.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Schematic illustration of mRNA sequence mapping workflow. Partial RNase T1 digests are performed in conjunction with LC–MS/MS analysis for automated oligoribonucleotide annotation and identification prior to sequence mapping.
Figure 2
Figure 2
Optimization of partial RNase T1 digests of mRNA. Total ion chromatograms of the partial T1 digests of Fluc mRNA. Twenty micrograms of RNA was incubated with varying amounts of immobilized RNase T1 equivalent to (top) 1.25 μL of RNase T1, (middle) 2.5 μL of RNase T1, and (bottom) 5 μL of RNase T1. All reactions were incubated for 10 min at 37 °C prior to LC–MS/MS analysis.
Figure 3
Figure 3
Reproducibility of partial RNase T1 digests of mRNA. (A) Total ion chromatograms of the partial RNase T1 digests of eGFP mRNA. Twenty micrograms of mRNA was incubated with 2.5 μL of immobilized RNase T1 for 10 min at 37 °C prior to LC–MS/MS analysis. The number of unique oligoribonucleotides and % sequence coverage are highlighted in each replicate. (B) Extracted ion chromatogram of three identified unique oligoribonucleotides from the LC–MS/MS analysis. The retention time and RSD across the replicates are shown for each oligoribonucleotide.
Figure 4
Figure 4
Mass spectrometry analysis of an oligoribonucleotide generated from the partial RNase T1 digest. (A) MS spectra of an oligoribonucleotide (5′UUCCC CAAUAUCACCAAUCUG3′-cP) from SARS CoV-2 spike protein mRNA. The corresponding monoisotopic m/z and charge states are highlighted. (B) Identified oligoribonucleotide fragment ions from the MS/MS spectra are shown for each charge state observed in the MS spectra.
Figure 5
Figure 5
RNA sequence mapping of mRNA therapeutics and long RNA. (A) Total ion chromatograms of the partial RNase T1 digests of RNA. In the RNase T1 digests, 20 μg of RNA was incubated with 2.5 μL of immobilized RNase T1 for 10 min at 37 °C. (B) Bar chart showing the % sequence coverage obtained for the complete RNase T1 digest, partial RNase T1 digest, and partial RNase T1 digest searched against a random sequence of the same size and GC content as for the target RNA. (C) Corresponding sequence coverage maps.
Figure 6
Figure 6
RNA sequence mapping of chemically modified mRNA. (A) Total ion chromatograms of the partial RNase T1 digests of Fluc mRNA and Fluc 5-methoxyU mRNA. Twenty micrograms of RNA was incubated with 1.25 μL of immobilized RNase T1 for 10 min at 37 °C prior to LC–MS/MS analysis. (B) Bar chart showing the % sequence coverage of the partial RNase T1 digest of Fluc mRNA (ORF sequence), chemically modified mRNA (ORF), and a random RNA sequence. (C, D) MS/MS spectra of the oligoribonucleotide CGGCUUCCGGGUGGUGCUGcP and the corresponding oligoribonucleotide where the uridines are replaced with 5-methoxyuridines. The corresponding fragment ions are highlighted, and those fragment ions specific to the 5-methoxyuridine oligoribonucleotide are highlighted in red.
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