sgRNA AND mRNA SEQUENCE MAPPING VIA ENDONUCLEASE DIGESTION AND LC-MS ANALYSIS, USING NOVEL INFORMATICS WORKFLOWS

Significance of the topic

Reliable and comprehensive mapping of RNA sequences—including single guide RNAs (sgRNAs) and long mRNAs—is essential for therapeutic development, quality control, and regulatory compliance. High-confidence sequence confirmation and modification mapping support product integrity, batch-to-batch consistency, and identification of unintended degradants or synthesis errors. Combining tailored endonuclease digestion strategies with high-resolution liquid chromatography–mass spectrometry (LC-MS) and dedicated informatics accelerates RNA characterization workflows and increases confidence in assignments for complex, modified RNA molecules.

Study objectives and overview

The presented study demonstrates an end-to-end LC-MS based workflow for sequence mapping and confirmation of sgRNA and long mRNA constructs. Key goals were to: achieve near-complete sequence coverage using complementary endonucleases; resolve isomeric/isobaric digestion products through data-independent fragmentation; and streamline experimental design, data processing and reporting using purpose-built informatics tools. The approach was validated on a 100-nt sgRNA standard and an ~1.8–2.0 kb firefly luciferase (FLuc) mRNA construct.

Methodology

Sample materials:

• Waters 100-mer sgRNA LC-MS standard encoding HPRT1, bearing 2′-O-methyl modifications on the first three 5′ and last three 3′ nucleotides and phosphorothioate internucleotide linkages at those modified positions.
• Custom in vitro transcribed FLuc mRNA with Cap1, ~1813 nt coding region and a poly(A) tail.

Endonuclease digestion strategy:

• Three enzymes were applied in parallel or complementary workflows: RNase T1 (G-specific cleavage), RapiZyme MC1 (cleaves 5′ of uridine with primary sites at [A/U/C]U and secondary sites at Cp[A/G]) and RapiZyme Cusativin (3′ cytidine specificity: primary Cp[A/U/G] and UpA motifs; secondary at [A/G]pU and UpU).
• Recombinant RapiZyme MC1 and Cusativin (RNase T2 family) produce longer overlapping digestion products due to unique specificity and miss-cleavage behavior, improving positional uniqueness of masses.

LC-MS acquisition and parameters:

• UHPLC: ACQUITY Premier System with ACQUITY Premier OST column (1.7 μm, 300 Å, 2.1 × 150 mm) at 70 °C; gradient 0→50% solvent B over 60 min; flow 0.4 mL/min.
• Mobile phases: A = 0.1% DIPEA, 1% HFIP in water (pH ≈8.5); B = 0.0375% DIPEA, 0.75% HFIP in 65% acetonitrile.
• Mass spectrometer: Xevo MRT (multi-reflectron TOF) QTOF operated in data-independent MSE mode; mass range m/z 340–4000; scan rate 0.5 s; low-energy CE 6 V; high-energy CE ramp 45–55 V.
• Typical resolving power achieved ≈100,000, enabling clear isotopic envelope separation even for high charge states.

Informatics and acceptance criteria:

• waters_connect platform with SYNTHETIC Library App and MAP Sequence App for digestion planning, in-silico product generation, automated matching and reporting.
• Identification tolerances: ±5 ppm mass error for precursor and fragments, ≥70% isotopic similarity, and minimum 50% sequence coverage per digestion product.
• Automated features include predicted coverage visualization, combination of multiple enzyme datasets, fragment ion verification, and comprehensive reporting for QC use.

Key results and discussion

Sequence coverage and digestion mapping:

• Using a multi-enzyme strategy (RNase T1, RapiZyme MC1, RapiZyme Cusativin) the 100-mer sgRNA achieved 100% sequence coverage with automated confirmation of sequence and terminal modifications.
• For the FLuc mRNA (~2000 nt), combined data from the three enzymes produced 98% linear sequence coverage despite complex chromatographic profiles.

Assignment confidence and fragmentation:

• Data-independent MS/MS (MSE) fragmentation resolved isomeric and isobaric digestion products by providing fragment ion evidence for sequence position assignments.
• Example: a 14-mer MC1 digestion product (U31:U44) was characterized across seven charge states, with full fragment coverage and clear isotopic resolution for the [M–8H]8– species.

Quantitative matching and accuracy:

• For the sgRNA MC1 digest, 77 predicted digestion products were matched within method tolerances; the average mass error for assigned MC1 products was reported as <1 ppm.
• Chromatograms showed the majority of detected peaks could be assigned to predicted digestion products, supporting robustness of the digestion planning and processing pipeline.

Benefits and practical applications

The integrated workflow provides several practical advantages:

• High-confidence sequence confirmation and modification localization for therapeutic RNAs and vaccine constructs.
• Ability to design complementary digestion strategies to maximize coverage while minimizing ambiguous assignments.
• Automation of processing and reporting supports routine use in QC and potentially manufacturing release testing under a compliance-ready informatics platform.
• High-resolution MS and MSE fragmentation reduce ambiguity from isomeric/isobaric digest products common in long or modified RNA molecules.

Used instrumentation

• UHPLC: Waters ACQUITY Premier System (binary) with TUV detection.
• Column: ACQUITY Premier OST Column, 1.7 μm, 300 Å, 2.1 × 150 mm.
• Mass spectrometer: Waters Xevo MRT QTOF MS (multi-reflectron time-of-flight).
• Enzymes: RNase T1 (Aspergillus oryzae), RapiZyme MC1 and RapiZyme Cusativin (recombinant RNase T2 family members).
• Informatics: waters_connect platform with SYNTHETIC Library App and MAP Sequence App.

Future trends and potential applications

Expected developments and practical directions include:

• Wider adoption of complementary, tunable RNases to enable routine mapping of increasingly modified therapeutic RNAs.
• Further integration of high-resolution DIA fragmentation and advanced informatics, including machine learning approaches, to automate ambiguous assignment resolution.
• Standardization of LC-MS-based RNA mapping protocols for regulatory submissions and lot-release testing in mRNA therapeutics and vaccine manufacturing.
• Expansion of software features for modification-aware in-silico digestion prediction and combined reporting across multiple analytical runs.

Conclusion

This work demonstrates that a coordinated approach—combining novel endonucleases (RapiZyme MC1 and Cusativin), high-resolution LC-MSE on the Xevo MRT QTOF, and purpose-built informatics—enables near-complete sequence mapping of both short sgRNAs and long mRNAs. The multi-enzyme strategy and DIA fragmentation markedly reduce assignment ambiguity and support automated, compliance-ready reporting suitable for research and quality environments.

Reference

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