Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA

Abstract

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1 H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing. The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13 C- and 15 Nlabeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1 H R1ρ experiments with both isotopically labeled and unlabelled RNA samples.