# Chemical reactivity of RNA and its modifications with hydrazine

## TL;DR

This study explores how hydrazine reacts with RNA and its modifications, offering a new method to distinguish RNA isomers using mass spectrometry.

## Contribution

The study introduces an aniline-free hydrazine-based method for isomer discrimination in RNA modifications.

## Key findings

- Hydrazine reacts with RNA modifications like pseudouridine and uridine, enabling isomer discrimination via MALDI-MS.
- Most modified nucleosides react quantitatively at low hydrazine concentrations without RNA cleavage.
- The method is applicable to native tRNA from E. coli and human cells, showing potential for RNA sequencing and MS workflows.

## Abstract

RNA modifications are essential for the regulation of cellular processes and have a key role in diseases such as cancer and neurological disorders. A major challenge in the analysis of RNA modification is the differentiation between isomers, including methylated nucleosides as well as uridine and pseudouridine. A solution is their differential chemical reactivity which enables isomer discrimination by mass spectrometry (MS) or sequencing. In this study, we systematically determine the chemical reactivity of hydrazine with RNA and its native modifications in an aniline-free environment. We optimize the conditions to achieve nearly full conversion of all uridines while avoiding RNA cleavage. We apply the conditions to native tRNAPhe which allows discrimination of pseudouridine and uridine by MALDI-MS. Furthermore, we determine the identity of the reaction product of hydrazine with various modified nucleosides using high resolution mass spectrometry and quantify the reaction yield in native tRNA from E. coli and human cells under various hydrazine conditions. Most modified nucleosides react quantitatively at lower hydrazine concentration while uridines do not decompose under these conditions. Thus, this study paves the way to exploit aniline-free hydrazine reactions in the detection of RNA modifications through MS and potentially even long-read RNA sequencing.

RNA modifications are essential for the regulation of cellular processes and have a key role in diseases, however, the differentiation between isomers of nucleosides remains a major challenge in the analysis of RNA modification. Here, the authors show a method with high potential for identification of modifications such as pseudouridine, 3-methylcytidine and others which offers advantages for both sequencing- and MS-based workflows.