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

Importance of the Topic

The use of short synthetic oligonucleotides and mRNA as therapeutic agents has expanded rapidly with applications in gene therapy and vaccines, notably mRNA COVID-19 vaccines. This growth drives the need for robust analytical quality control at multiple stages from research and development to production

Objectives and Study Overview

This resource guide presents end-to-end workflows for the analysis of short nucleic acids and mRNA. It aims to outline methodologies for assessing sample size, purity, thermal stability, 5 prime capping and 3 prime poly A tail characterization across various instrumental platforms

Methodology and Instrumentation

• Workflow 1 Short Nucleic Acid Analysis by Capillary Electrophoresis using fragment analyzers and Oligo Pro II systems for high resolution sizing of small RNAs and ssDNA oligos
• Workflow 2 mRNA Purity and Size Assessment by capillary electrophoresis with Fragment Analyzer and TapeStation systems and UV Vis spectroscopy with Cary 3500 and BioTek microplate readers for concentration and purity checks
• Workflow 3 mRNA 5 prime capping efficiency and 3 prime poly A Sequence analysis via LC MS using Infinity II BioLC systems, oligonucleotide columns, RNase digestion and Q TOF detection followed by BioConfirm software processing

Main Results and Discussion

The described platforms deliver high throughput and accurate sizing of nucleic acid fragments from below 60 bases to several kilobases. Capillary electrophoresis systems enable unattended processing of up to three 96 well plates. UV Vis and fluorescence measurements provide rapid concentration and purity data. LC MS workflows achieve precise mapping of capping structures and poly A length variants

Benefits and Practical Applications

• Ensures reliable quality control throughout R D and production of RNA therapeutics
• Optimizes purification protocols and IVT workflows by detailed characterization of caps and tails
• Supports high throughput screening in both research and QC laboratories with modular automation

Instrumentation

• Agilent Fragment Analyzer 5200, 5300, 5400 systems and TapeStation 4200
• Agilent Oligo Pro II with 12 24 or 96 capillary arrays
• Agilent Cary 3500 UV Vis spectrophotometer and BioTek Synergy H1 microplate reader with Take3 plate
• Agilent 1290 Infinity II Bio LC, PLRP S and AdvanceBio columns, 6545XT Q TOF MS
• Agilent ProSize, TapeStation Analysis and MassHunter BioConfirm software

Future Trends and Opportunities

Advances will focus on further automation, miniaturization and integration of analytical platforms. Machine learning driven data analysis may enhance detection of subtle sequence and structural variants. New chemistries and detection modalities could expand capabilities for real time monitoring and single cell RNA analysis

Conclusion

The comprehensive workflows outlined here provide robust solutions for short oligonucleotide and mRNA analysis from R D through QC and production. By combining capillary electrophoresis, spectrophotometry and LC MS techniques, laboratories can achieve high throughput, accuracy and detailed characterization of critical quality attributes

References

1. Agilent Technologies 2022 Comparison of Small RNA Analysis using the Agilent Bioanalyzer and Fragment Analyzer Systems Technical Overview 5994 4860EN
2. Agilent Technologies 2021 Performance Characteristics of the Agilent Oligo Pro II System Technical Overview 5994 3667EN
3. Agilent Technologies 2022 Nucleic Acid Analysis for Sample Quality Assessment Using the Agilent Fragment Analyzer Systems Application Compendium 5994 2813EN
4. Agilent Technologies 2019 Single Guide RNA Quality Assessment with the Agilent 5200 Fragment Analyzer System Application Note 5994 0523EN
5. Agilent Technologies 2021 Accurate Assessment of Oligonucleotide Purity Brochure 5994 0421EN