Oligonucleotide and mRNA Analysis Resource Roundup

Oligonucleotide therapeutics keep expanding in complexity, and the analytics expectations are rising right alongside them. ASOs, siRNA, and mRNA all bring familiar requirements (identity, purity, impurity characterization, termini confirmation, modification control), but they also add new pressure on throughput, method robustness, and data defensibility. For many teams, the limiting factor is no longer only the instrument method. It is how quickly you can turn data into consistent, reviewable answers.

This post brings together the 2025 content we produced across intact oligo LC-MS, impurity quantitation, and mRNA mapping. There’s also some useful resources for looking at the future challenges for this field, and how we can help you solve them.

If you only do one thing, register for the live mRNA mapping webinar on April 22, 2026 sequence confirmation workflows toward practical, automated execution.

Need a primer on oligonucleotides?

This review article Therapeutic Oligonucleotides: State of the Art, is one of the best reviews on oligonucleotide therapeutics covering the major modalities and mechanisms (antisense gapmers, steric-block/splice-switching oligos, and siRNA), along with key chemistry concepts, delivery strategies, clinical progress, and the pharmacology considerations that shape successful development.

Start here: register for the on demand webinar recording from April 22, 2026

April 22, 2026 | 10:00 London • 11:00 Berlin • 13:00 Dubai | Live and on-demand
Speaker: Jessica Dale, PhD Student in Oligonucleotide Analysis, University of Sheffield
This one-hour seminar highlights a fully automated 2D LC-MS workflow that integrates online partial RNase T1 digestion to achieve 93 to 99 percent mRNA sequence coverage in under 60 minutes, with automated multi-attribute monitoring. It is highly relevant whether you are building capability in R&D or thinking about how mapping can scale toward routine decision-making.

If your team is exploring broader oligonucleotide and mRNA workflows and Thermo Scientific’s supporting technologies, these pages are useful for deeper context:

mRNA Vaccine and Therapeutics Research & Development | Thermo Fisher Scientific
Oligonucleotide Analysis | Thermo Fisher Scientific

On-demand webinars from discovery to QC with insights from experts in academia and industry

These sessions are part of the Advancements and Optimisation of Analytical Technology for Oligonucleotide Analysis Series, which includes additional related content. This is a good internal share link because it supports self-serve learning across different roles and experience levels.

mRNA mapping and MAM-style thinking

Professor Mark Dickman from the University of Sheffield showcases a workflow designed to enhance the precision and efficiency of mRNA analysis. The approach combines Multi-Attribute Monitoring concepts with online partial RNase T1 digestion and LC-MS.

Watch the webinar: Multi-Attribute Monitoring of mRNA Medicines

Read the case study: Blueprint for QC-Ready mRNA Mapping with Thermo Fisher and the University of Sheffield

Advanced characterization resources: fragmentation control, chemistry research, and emerging assay concepts

As oligo modalities mature, characterization problems shift. You often see more emphasis on fragmentation quality, adduct management, and new routes to higher throughput sequence confirmation. These resources are useful when you are past the basics and tuning workflows for confidence, speed, or scalability.

This peer reviewed study Characterisation of small RNA-based therapeutics and their process impurities by fast and sensitive liquid chromatography high resolution mass spectrometry, with our partners at the National Institute for Bioprocessing Research and Training (NIBRT) walks through a fast, sensitive amine-based ion-pair RP-LC–HRMS/MS workflow for chemically modified ss-ASOs, including practical strategies to reduce adducting and in-source artifacts while enabling confident impurity characterization and sequence confirmation.

Low-q CID for oligonucleotides: improving fragmentation behaviour for sequencing confidence

Thermo Scientific Orbitrap Tribrid mass spectrometers combine the best of Orbitrap analysers and linear ion trap mass analysers, and we have seen some amazing advances with this technology applied to oligonucleotide analysis. Gain a competitive edge with the most advanced MS/MS sequencing and see how it helps solve real-world challenges in the lab.

Low-q CID

Watch now: Teach Me in 10; video overview on low-q CID.

Low q CID is an RF ion trap resonant collision induced dissociation technique for oligonucleotide anions that activates precursor ions at reduced Mathieu q values to suppress undesirable second-generation fragment ions.

Thermo Scientific scientists developed this approach to solve a long-standing problem in oligonucleotide MS, where conventional fragmentation often creates too much unintended breakdown to support clear and confident interpretation. This matters because the optimized low q CID approach reduces unwanted fragmentation, making oligonucleotide mass spectra much easier to interpret and sequence assignments much more reliable; this is particularly true for longer sequences where the push to more optimised fragmentation allows for lower abundance high mass fragment ions to become routinely measurable.  

Learn more by downloading the poster: Optimized Fragmentation of Oligonucleotides Suppresses Undesired Fragmentation Products and Enables Confident Sequence Assignment.

The most advanced mass spectrometry technologies

This study, ‘Key RNA-binding domains in the La protein establish tRNA modification levels in Trypanosoma brucei’ shows how the Orbitrap Ascend BioPharma Tribrid mass spectrometer and Thermo Scientific Compound Discoverer software can help researchers move beyond detection to confidently map biologically important tRNA modification changes at scale, revealing how RNA binding by the La protein reshapes the epitranscriptomic landscape in Trypanosoma brucei.

By combining high resolution LC MS/MS with streamlined data analysis, the workflow enabled sensitive, modification level profiling that captured both the sharp rise in queuosine and broader shifts across the tRNA modification network, giving researchers a clearer path to biologically meaningful insight

To learn more about what Compound Discoverer software can bring to your lab, start here: Compound Discoverer Software | Thermo Fisher Scientific.

You can also learn more about the Orbitrap Tribrid Mass Spectrometer here: Orbitrap Tribrid Mass Spectrometers | Thermo Fisher Scientific.

Research advances in oligo analysis chemistry

If you are re-evaluating method design choices, including mobile phase and ionization behaviour, this paper, Liquid chromatography – high resolution mass spectrometry analysis of intact ribonucleic acid through hydrogen bonding of alkylamines highlights ongoing research directions in oligo analysis chemistry.

New fragmentation models for prediction algorithms

This JASMS publication, “Prediction of collision-induced dissociation spectra of deprotonated oligonucleotides”, describes a computational approach for predicting CID MS/MS fragmentation patterns for oligonucleotides, with the goal of making sequence confirmation and spectral interpretation faster and more confident.

Our team is actively tracking and incorporating leading advances like this into our roadmap, so customers can expect continued progress in how Thermo Scientific BioPharma Finder software supports oligonucleotide MS/MS interpretation and reporting.

This is the BioPharma Finder product page if you want to explore capabilities more broadly.

tRNA characterization via intact mass analysis

tRNA systems are useful stress tests for intact mass workflows because of their dense modification patterns. Even if you are not working directly on tRNA, the strategies can inform how you think about deconvolution robustness and intact mass interpretation and help you identify low level impurities with accuracy and confidence.

Download the poster: Comprehensive Characterization of tRNA by Intact Mass Analysis

Four articles to support method decisions on oligonucleotide therapeutics

If you are building a rationale for workflow choices, these papers provide useful framing across CQAs, source-condition impacts, and mechanistic issues that can affect interpretation.

1. Camperi, J., Chatla, K., Freund, E., Galan, C., Lippold, S. and Guilbaud, A. (2025) ‘Current analytical strategies for mRNA-based therapeutics’, Molecules, 30(7), 1629. doi:10.3390/molecules30071629.
2. Dale, J.S., Welbourne, E.N., Evans, C.A., Minshull, T.C., Schwahn, A.B., Rupprecht, F., Cook, K., Loveday, K.A., Kis, Z. and Dickman, M.J. (2026) ‘Automated Online Direct mRNA Sequence Mapping Using Partial RNase T1 Digests’, Analytical Chemistry. doi: 10.1021/acs.analchem.5c08110.
3. Glenister, M.A., Mistarz, U., Cook, K., Stephenson, J.L. and Dickman, M.J. (2025) ‘Optimisation of heated electrospray ionisation parameters to minimise in-source generated impurities in the analysis of oligonucleotide therapeutics’, Rapid Communications in Mass Spectrometry, 39(13), e10033. doi:10.1002/rcm.10033.
4. Peronin, S., Malburet, C., Daher-Hassan, C., Picard, C., Willemin, T., Costamagna, F., Even, L., Eynard, T., Bensaid, F., Fertier-Prizzon, S. and Francois-Heude, M. (2025) ‘Analytical approach for identification and mechanistic insights into mRNA-lipid adduct formation’, Molecular Therapy – Nucleic Acids, 36(3), 102684. doi:10.1016/j.omtn.2025.102684.

A note on what’s coming next: conjugates and higher complexity modalities

A theme that keeps surfacing is that new modalities do not replace old analytical questions. They stack new questions on top of them. Antibody–oligonucleotide conjugates are a good example because they combine oligo characterization requirements with bioconjugate heterogeneity and linker chemistry considerations.

If you want an outside perspective on why this modality matters, this Nature Biotechnology review Now with oligos: antibody–oligonucleotide conjugates are the new drug modality to watch is a useful starting point:

Closing: choose the path that matches your role, then go deeper.

Explore our solution pages for additional context and resources:

• Nucleic Acid Therapeutic Development | Thermo Fisher Scientific
• Oligonucleotide Analysis | Thermo Fisher Scientific
• Pharma & Biopharma Learning Center | Thermo Fisher Scientific

Visit us on LinkedIn: #OligonucleotideAnalysis #mRNAAnalysis #MassSpectrometry #Biopharma