INVESTIGATING CYCLIC ION MOBILITY AS A MEANS TO BOOST COVERAGE IN THE TOP-DOWN SEQUENCING OF SYNTHETIC GUIDE RNAS

Significance of the Topic

The rise of CRISPR-based gene editing has placed synthetic guide RNAs (sgRNAs) at the forefront of therapeutic research. Accurate sequence confirmation of sgRNAs is critical for ensuring specificity and safety in cell and gene therapy workflows. Traditional mapping methods often involve enzymatic digestion and lengthy liquid chromatography–mass spectrometry (LC–MS) runs, leading to time-consuming workflows.

Objectives and Study Overview

This work evaluates the application of cyclic ion mobility separation to enhance sequence coverage in top-down mass spectrometry of synthetic sgRNAs. Four oligonucleotides were compared: a full-length 100-nucleotide sgRNA and truncated 25, 50, and 75 nucleotide fragments. The goal was to determine whether mobility separation reduces spectral congestion and increases terminal fragment identification, thereby improving sequence coverage.

Methodology and Instrumentation

RNA samples were introduced by direct infusion in HDMSE mode and, for intact mass analysis, separated by ACQUITY UPLC Premier with an oligonucleotide BEH C18 column (1.7 µm, 2.1 × 100 mm) at 60 °C. Mobile phases comprised triethylamine (TEA)/hexafluoroisopropanol (HFIP) buffers in water and methanol/water.

All fragmentation experiments were conducted on the SELECT SERIES Cyclic IMS time-of-flight mass spectrometer (Rp = 100 000 FWHM) operated in single-pass ion mobility mode. Product ions from collision-induced dissociation (CID) were separated into 3–5 mobility fractions. Data were extracted with Driftscope v3.0 and processed using the CONFIRM sequence application in the waters_connect suite. Key processing parameters included mass error ≤ 15 ppm, intensity cutoff ≥ 10 counts, and isotope similarity ≥ 70 %. Only terminal fragment ions were matched.

Main Results and Discussion

Comparison of sequence coverage with and without ion mobility demonstrated clear benefits, particularly for longer oligonucleotides:

• 25 mer: Matched ions increased from 101 to 111; coverage remained at 100 % in both cases.
• 50 mer: Ions rose from 125 to 146; coverage improved from 96 % to 100 %.
• 75 mer: Ions rose from 135 to 186; coverage jumped from 67 % to 96 %.
• 100 mer: Terminal ions identified increased to yield coverage from 50 % (without mobility) to 74 % (with mobility).

The largest improvements occurred with the 75 mer and 100 mer, where spectral congestion is greatest. Ion mobility separation reduced peak overlap in each fraction, simplifying assignment and boosting terminal fragment detection.

Benefits and Practical Applications

Cyclic ion mobility offers rapid, gas-phase separation of fragment ions without adding chromatographic steps. This approach:

• Enhances sequence coverage in top-down workflows.
• Reduces spectral complexity and improves confidence in fragment assignment.
• Shortens overall analysis time by eliminating digestion and extended LC gradients.

Future Trends and Opportunities

Opportunities to further increase coverage include matching internal fragments and integrating complementary fragmentation methods such as electron capture dissociation. Advances in software automation and real-time mobility-enabled sequencing could enable higher throughput characterization of therapeutic oligonucleotides and complex RNA assemblies.

Conclusion

The study demonstrates that cyclic ion mobility separation on the SELECT SERIES Cyclic IMS instrument significantly improves sequence coverage in top-down MS of synthetic guide RNAs. The most pronounced gains were observed for longer oligonucleotides, where spectral congestion is most severe. High-resolution TOF analysis combined with mobility fractionation represents a powerful platform for rapid, detailed characterization of sgRNA therapeutics.

Reference

1. Avtonomov D.M., Polasky D.A., Ruotolo B.T., Nesvizhskii A.I. Anal. Chem. 2017, 90(3), 2369–2375.
2. Shaw J.B., Cooper-Shepherd D.A., Hewitt D., Wildgoose J.L., Beckman J.S., Langridge J.I., Voinov V.G. Anal. Chem. 2022, 94(9), 3888–3896.