# Exploring RNA G‐Quadruplex Stability in the Gas Phase: Insights from Native Mass Spectrometry

## TL;DR

This study uses mass spectrometry to explore how RNA G-quadruplex structures remain stable in the gas phase after desolvation.

## Contribution

The study reveals that RNA G-quadruplex stability in the gas phase correlates with their stability in solution and is affected by net charge and central cations.

## Key findings

- RNA G-quadruplexes with higher solution stability maintain higher stability in the gas phase regardless of ion charge.
- Coulombic repulsion weakens hydrogen bonding and cation-nucleobase interactions in gaseous RNA G-quadruplexes with higher net charge.
- Covalent bond cleavage occurs at lower energies than strand separation for lower net charge RNA G-quadruplexes with K+ as central cations.

## Abstract

Native mass spectrometry (MS) of ribonucleic acids (RNA) is a developing technique for the study of RNA structure and interactions that can provide important information on binding stoichiometry and binding motifs. However, the stabilities of key structural elements of RNA, including G‐quadruplex structures, during and after desolvation are not well understood. Efforts to improve our understanding of RNA structures in the gas phase are complicated by the fact that the most common technique for transferring RNA from solution to the gas phase, electrospray ionization (ESI), typically produces ions with a relatively wide distribution of net charges. Here, we have studied two tetramolecular RNA G‐quadruplexes with very different stabilities in solution by native MS. For both K+ and +NH4 as central cations, we found that the ratio of quadruplex to monomer ions in the ESI spectra was higher for the RNA G‐quadruplex that exhibited higher stability in solution, regardless of ion net charge. These data for native MS of RNA in negative ion mode contrast with data from native MS of proteins in positive ion mode, for which lower and higher net charge is often associated with folded and unfolded structures, respectively. We next probed the stabilities of the gaseous RNA G‐quadruplex ions using collisionally activated dissociation (CAD). For quadruplex ions with K+ as central cations, we observed a steady decrease in stability with increasing net charge, consistent with Coulombic repulsion weakening the hydrogen bonding networks and the interactions between the central cations and the nucleobases forming the central channel of the quadruplex. Importantly, for quadruplex ions with the same net charge and for both K+ and +NH4 as central cations, the RNA G‐quadruplex that exhibited higher stability in solution also demonstrated higher stability in the gas phase. Furthermore, we demonstrate that for quadruplexes with lower net charge and K+ as central cations, covalent bond cleavage occurs at lower energies available for dissociation than strand separation.

This study explores RNA G‐quadruplex structures using native mass spectrometry to better understand their stability during desolvation and in the gas phase. Although increasing net charge reduces quadruplex stability due to Coulombic repulsion, even highly charged quadruplex ions do not spontaneously dissociate. Notably, for quadruplexes with lower net charge, covalent bond cleavage occurs at lower energies than strand separation.© 2026 WILEY‐VCH GmbH