Synthetic modified oligonucleotides analysis using a matrix-assisted laser desorption/ionization digital-ion-trap mass spectrometer (MALDI-DIT-MS)

Importance of the Topic

Matrix-assisted laser desorption/ionization coupled with a digital ion trap mass spectrometer (MALDI-DIT-MS) provides a rapid and simplified platform for the molecular characterization of synthetic modified oligonucleotides. As nucleic acid therapeutics expand, there is a growing need for high-throughput methods to confirm molecular weight, sequence integrity, site-specific modifications and conjugations, avoiding lengthy liquid chromatography workflows.

Objectives and Study Overview

This work introduces a streamlined MALDI-DIT-MS approach to analyze three classes of therapeutic oligonucleotides: single-stranded DNA (mipomersen), single-stranded RNA (patisiran) and double-stranded RNA (vutrisiran). The goals were to demonstrate sensitive mass measurement, confirm base sequence and detect specialized modifications, including triantennary GalNAc conjugates, using a single instrumentation platform.

Methodology and Instrumentation

Samples were prepared by mixing analyte solutions in acetonitrile/water with ammonium hydrogen citrate matrix solutions. A 1:1 (v/v) mix was deposited on a target plate and analyzed by MALDImini-1 in positive ion mode with raster scanning. Mass analysis conditions varied by sample type, optimizing laser settings, detector voltage and delay times. Fragmentation (MS/MS and MS3) was performed for sequence confirmation and detection of modification-specific ions.

Key Findings and Discussion

• Single-stranded DNA (7177 Da) and RNA (6764 Da) were detected as [M+H]+ ions with high sensitivity.
• Double-stranded RNA (16,345 Da total) generated distinct [M+H]+ and multicharged species.
• MS/MS fragmentation mapped backbone cleavages (a, b, w, y ions) to confirm sequence and locate 2’-O-methyl and fluorinated nucleotides.
• Triantennary GalNAc conjugates produced diagnostic triplet peaks 203 Da apart, and MS3 spectra validated terminal modification sites.

Benefits and Practical Applications

The MALDI-DIT-MS method offers:

• Rapid molecular weight verification without chromatographic separation.
• Direct sequence analysis through multi-stage MS.
• Sensitive detection of base modifications and ligand attachments.
• Reduced sample consumption and simplified workflow for quality control.

Future Trends and Applications

Advances may include automated sample spotting, integration with microarray formats and expanded libraries of matrices for tailored sensitivity. Further development of high-resolution DIT analyzers and on-target digestion protocols could extend applications to longer oligonucleotides and complex conjugates.

Conclusion

This study demonstrates that a single MALDI-DIT-MS system can perform comprehensive molecular weight, sequence and modification analyses for therapeutic oligonucleotides, streamlining quality control and structural verification workflows.

Reference

• McLuckey SA, Journal of the American Society for Mass Spectrometry, 1992, 3:60–70