Rapid Analysis of mRNA 5' Capping with High Resolution LC/MS

Importance of the Topic

The 5’ cap structure is a key determinant of mRNA stability and translational efficiency. In mRNA therapeutics and vaccines, such as those developed against SARS-CoV-2, precise quantification of capping is essential to ensure product quality, potency, and regulatory compliance.

Objectives and Study Overview

This work presents a rapid liquid chromatography–mass spectrometry (LC/MS) method for detailed analysis of mRNA 5’ capping. It streamlines sample preparation through the use of thermostable RNase-H and evaluates both co-transcriptional (ARCA) and enzymatic (Vaccinia) capping strategies.

Methodology and Instrumentation

• mRNA Synthesis: In vitro transcription of a ~3.8 kb template with T7 RNA polymerase, DNase I treatment, LiCl precipitation and cleanup.
• Site-Directed Cleavage: Design of chimeric 2′-O-methyl RNA/DNA probes targeting 5′ regions (40 nt and 50 nt). Comparison of conventional 37 °C RNase-H cleavage vs 50 °C cleavage with thermostable RNase-H to eliminate a separate annealing step.
• Chromatography: Ion-pair reversed-phase separation on an Agilent AdvanceBio Oligonucleotide column (2.1×50 mm, 2.7 µm, 120 Å) using dibutylamine/HFIP gradients at 0.4 mL/min and 50 °C.
• Mass Spectrometry: Agilent 6545XT AdvanceBio LC/Q-TOF in negative mode (400–3200 m/z), high sensitivity; data processed with MassHunter BioConfirm for deconvolution and quantitation.

Main Findings and Discussion

Integration of thermostable RNase-H at 50 °C reduced total sample preparation and analysis time to ~75 minutes while avoiding off-target cleavage seen with high-GC probes at 37 °C. LC/MS analysis achieved baseline separation of uncapped diphosphate/triphosphate oligonucleotides from capped species with <20 ppm mass accuracy.

• ARCA Co-Transcriptional Capping: Achieved 91.3 ± 1.8% Cap 0 efficiency; subsequent methylation to Cap 1 yielded 90.6 ± 1.5% conversion.
• Vaccinia Enzymatic Capping: Standard protocol produced only 9.1 ± 0.6% Cap 0; optimizing enzyme-to-mRNA ratio (0.25–0.5× input mRNA) increased capping to 17.7 ± 1.8% and 32.9 ± 0.4%, respectively.
• Transcriptional Slippage: Detection of non-templated +G variants illustrated the sensitivity of LC/MS to sequence heterogeneity.

Benefits and Practical Applications

This high-resolution, rapid workflow enables sensitive quantitation of mRNA capping, supporting process optimization and quality control in biopharmaceutical development. The streamlined protocol and flexible probe design facilitate broad applicability to diverse mRNA sequences without reliance on affinity enrichment.

Future Trends and Applications

Advances may include automated, high-throughput sample handling, refined probe design to further reduce off-target interactions, and expansion to analyze additional RNA modifications. Adoption in regulated QC environments will accelerate mRNA therapeutic manufacturing.

Conclusion

The described LC/MS method employing thermostable RNase-H offers a fast, robust platform for the analysis of mRNA 5’ capping, enhancing process control and ensuring quality of emerging nucleic acid-based therapies.

Instrumentation

• Agilent 1290 Infinity II LC with diode array detector.
• Agilent AdvanceBio Oligonucleotide column (2.1×50 mm, 2.7 µm, 120 Å).
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer.

Reference

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