Poster Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

Ion pairing-free mass spectrometry analysis of RNA oligonucleotides: the quantification of mRNA capping efficiency and CRISPR/Cas13 cleavage as case studies (#18)

Enzo Huang 1 2 , Roland Calvert 1 , Vita Levina 1 2 , Gavin Knott 1 , Chen Davidovich 1 2
  1. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
  2. RNA Mass Spectrometry Platform, Monash Proteomics & Metabolomics Platform, Monash University, Clayton, VIC, Australia

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a critical analytical tool for characterising RNA oligonucleotides, yet the current approaches often heavily rely on ion-pairing reagents. Ion-pairing reagents can potentially introduce ion suppression and contamination to the LC-MS/MS systems. Therefore, the usage of ion pairing reagents remained a major bottleneck for introducing RNA mass spectrometry workflows into conventional mass spectrometry facilities.

Here, we describe a hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) method that eliminates the need for ion-pairing agents while maintaining relatively high sensitivity for RNA analysis. We demonstrate the versatility of this approach through two applications: (1) assessing the capping efficiency of in vitro transcribed mRNA and (2) monitoring RNA cleavage by the CRISPR-associated protein Cas13a.

For mRNA therapeutics, 5' capping is essential for stability and translational efficiency. While some sequencing methods can provide indirect information about capping, they cannot precisely identify cap structures or accurately quantify capping efficiency. We show that the ion-pairing-free application of HILIC-MS/MS can accurately quantify capping efficiency by directly detecting uncapped, Cap-0, and Cap-1 mRNA species.

Cas13 is a group of RNA-guided RNA-targeting enzymes that attract much interest given their potential in diagnostic and therapeutic applications. Yet, characterising their mechanism of action has been challenging, partially due to the small size of the product and its heterogeneity. Here we show that HILIC-MS/MS, without ion pairing reagents, provides detailed insights into Cas13a cleavage site preferences, the obtained product. We show that HILIC-MS/MS not only precisely measured the mass of the cleavage products, but also achieved chromatographic separation with the mass difference of a single phosphate group.

This study establishes a simplified yet powerful alternative for the use of ion pairing reagents for the mass spectrometry of RNA oligonucleotides. The developed HILIC-MS/MS approach would enable conventional mass spectrometry facilities access to RNA mass spectrometry workflows, offering practical advantages in a shared instrument environment.