RNA structure determination by NMR.

This chapter reviews the methodologies for RNA structure determination by liquid-state nuclear magnetic resonance (NMR). The routine production of milligram quantities of isotopically labeled RNA remains critical to the success of NMR-based structure studies. The standard method for the preparation of isotopically labeled RNA for structural studies in solution is in vitro transcription from DNA oligonucleotide templates using T7 RNA polymerase and unlabeled or isotopically labeled nucleotide triphosphates (NTPs). The purification of the desired RNA can be performed by either denaturing polyacrylamide gel electrophoresis (PAGE) or anion-exchange chromatography. Our basic strategy for studying RNA in solution by NMR is outlined. The topics covered include RNA resonance assignment, restraint collection, and the structure calculation process. Selected examples of NMR spectra are given for a correctly folded 30 nucleotide-containing RNA.