The impact of RNA chemical probing reagents on RNA-binding proteins.

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 flexible RNA nucleotides. The highly reactive nature of the chemical probes means that they can also react with nucleophilic amino acid side chains, subsequently affecting protein-RNA binding events. We combine molecular dynamics (MD) simulations, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and nuclear magnetic resonance (NMR) experiments to demonstrate that commonly used RNA chemical probes interact with protein amino acids and that this interaction alters protein-RNA binding through binding assays. We discuss the implications of this phenomenon in elucidating the protein-RNA interaction interface using chemical probing experiments.