Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R_1ρ relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2-6 weeks and moderate understanding of NMR and RNA structure. We recently applied the MCSF methodology to elucidate the excited state of microRNA 34a targeting the SIRT1 mRNA and use this example to demonstrate the analysis. The protocol comprises the following steps: (i) determination of the secondary structure of the excited state from RD chemical shift data, (ii) design of trapped excited state RNA, (iii) validation of the excited state structure by NMR, and (iv) MCSF analysis comparing the chemical shifts of the trapped excited state with the RD-derived chemical shift differences. MCSF enables observation of the short-lived RNA structures, which can be functionally and structurally characterized by entrapment.