End-To-End Workflow Solutions for Short Nucleic Acids and mRNA Analysis

Importance of the Topic

Short synthetic oligonucleotides and mRNA-based therapeutics have become central to gene therapy and vaccine development. Reliable analytical workflows are essential to characterize critical quality attributes—such as sequence integrity, size distribution, purity, 5′ capping efficiency, and poly(A) tail length—that directly impact biological activity and stability.

Objectives and Study Overview

This guide outlines end-to-end analytical solutions for nucleic acid and mRNA workflows, including:

• High-resolution sizing and purity assessment of short RNAs (≤200 nt) by capillary electrophoresis.
• Comprehensive mRNA QC at multiple stages—plasmid template, IVT product, poly(A) tail, final vaccine—using CE, TapeStation, and UV-Vis spectrophotometry.
• Quantification of 5′ capping efficiency and detailed poly(A) tail profiling by LC-MS methodologies.

Methodology

• Capillary Electrophoresis (CE): Automated, multi-capillary systems separate single-stranded DNA and RNA by size under denaturing conditions, with UV detection for dye-free purity analysis.
• UV-Vis and Microplate Detection: Low-volume and high-throughput measurement of nucleic acid concentration and purity ratios at 260 nm, using both cuvette-based and microplate formats.
• LC-MS for Capping and Poly(A) Analysis: Enzymatic digestion and magnetic-bead enrichment workflows enable intact and fragment-level analysis of 5′ cap structures and heterogeneous poly(A) tails on high-resolution Q-TOF platforms.

Used Instrumentation

• Agilent Fragment Analyzer systems (5200, 5300, 5400) and Oligo Pro II CE platforms (12/24/96 capillaries)
• Agilent 4200 TapeStation
• Agilent Cary 3500 UV-Vis spectrophotometer; BioTek Synergy H1 multimode reader with Take3 microvolume plates; BioTek Epoch 2
• Agilent 1290 Infinity II Bio LC with AdvanceBio Oligonucleotide columns
• Agilent 6545XT AdvanceBio LC/Q-TOF mass spectrometer with MassHunter BioConfirm software
• Data analysis: ProSize, TapeStation Analysis, Cary UV Workstation, Oligo Pro II Software, BioTek Gen6

Main Results and Discussion

1. CE workflows deliver single-nucleotide resolution and high-throughput screening of short RNAs, ensuring accurate size and purity metrics.
2. Automated TapeStation and Fragment Analyzer systems enable seamless switching between DNA and RNA analyses, supporting multiple QC checkpoints in mRNA production.
3. UV-Vis and fluorescence microplate assays provide rapid, robust quantification and contaminant detection, scalable to 384-well formats.
4. LC-MS strategies achieve detailed mapping of 5′ cap incorporation and poly(A) tail heterogeneity, critical for predicting mRNA translation efficiency and stability.

Benefits and Practical Applications

• Standardized, scalable workflows reduce hands-on time and increase data consistency across R&D, QC, and manufacturing.
• Flexible throughput options accommodate single-sample analyses up to high-density plate formats.
• Comprehensive characterization supports regulatory compliance and accelerates development of nucleic acid–based therapies and vaccines.

Future Trends and Opportunities

• Integration of real-time, inline analytical modules for continuous process monitoring.
• Advanced informatics pipelines to correlate multi-modal QC data with functional outcomes.
• Development of multiplexed assays for simultaneous evaluation of multiple quality attributes in a single run.

Conclusion

The presented workflows offer robust, high-throughput analytical solutions for short nucleic acids and mRNA products. By combining capillary electrophoresis, UV-Vis spectrophotometry, and advanced LC-MS techniques, laboratories can achieve precise QC of critical quality attributes, thereby accelerating the development and production of nucleic acid therapeutics and vaccines.

References

1. Comparison of Small RNA Analysis using the Agilent Bioanalyzer and Agilent Fragment Analyzer Systems; Agilent Technologies Technical Overview, 5994-4860EN (2022).
2. Performance Characteristics of the Agilent Oligo Pro II System; Agilent Technologies Technical Overview, 5994-3667EN (2021).
3. Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems; Agilent Technologies Application Compendium, 5994-2813EN (2022).
4. Single-Guide RNA Quality Assessment with the Agilent 5200 Fragment Analyzer System; Agilent Technologies Application Note, 5994-0523EN (2019).
5. Accurate Assessment of Oligonucleotide Purity; Agilent Technologies Brochure, 5994-0421EN (2021).
6. Best Practices for Analysis of IVT mRNA Using the Agilent Fragment Analyzer Systems; Agilent Technologies Technical Overview, 5994-6568EN (2023).
7. Best Practices for Analysis of In Vitro Transcribed (IVT) mRNA Using the Agilent Fragment Analyzer Systems; Agilent Technologies Technical Overview, 5994-5927EN (2023).
8. Quality Control in IVT RNA Workflow using Agilent TapeStation Systems; Agilent Technologies Technical Overview, 5994-4882EN (2022).
9. Best Practice for Nucleic Acid Thermal Stability Measurements Using the Cary 3500 UV-Vis Spectrophotometer; Agilent Technologies White Paper, 5994-4028EN (2022).
10. Assessment of Long IVT mRNA Fragments with the Agilent 5200 Fragment Analyzer System; Agilent Technologies Application Note, 5994-0878EN (2019).
11. Benefits of Quality Control in the IVT RNA Workflow Using the Agilent 5200 Fragment Analyzer System; Agilent Technologies Application Note, 5994-0512EN (2023).
12. Analyzing Poly(A) Tails of IVT RNA with the Agilent Fragment Analyzer System; Agilent Technologies Technical Overview, 5994-5325EN (2022).
13. Rapid Analysis of mRNA 5' Capping with High-Resolution LC/MS; Agilent Technologies Application Note, 5991-3984EN (2021).
14. Analysis of mRNA Poly-A Sequence Variants by High-Resolution LC/MS; Agilent Technologies Application Note, 5994-3005EN (2021).