RNA chemical probing experiments are a broadly used method for revealing the structure of RNA, as well as for identifying protein binding sites. This is beneficial for expanding our understanding of biological processes governed by protein-RNA complex interactions, as well as facilitating the identification of complex inhibiting molecules. The reagents commonly used in chemical probing experiments are highly reactive, methylating or acylating atoms of flexible RNA nucleotides. The highly reactive nature of the chemical probes means that they can also react with nucleophilic amino acid side chains, and subsequently affect protein-RNA binding events. We combine MD simulations, MALDI-MS, and NMR experiments to show that commonly used RNA chemical probes react with protein amino acids, and demonstrate that this effect alters protein-RNA binding interactions through binding shift assays. We discuss the implications of this phenomenon in elucidating protein-RNA interactions, and also demonstrate this as a useful means for identifying surface-exposed protein residues, omitting the need for extensive mutagenesis experiments that are critical for RNA interactions.

RNA Chemical Probing Reagents and Protein Amino Acids: a Double-Edged Sword