Nuclear Magnetic Resonance Spectroscopy of RNA

Abstract

High-resolution structure has been a missing element in our understanding of RNA function. Advances in crystallographic and nuclear magnetic resonance (NMR) methodologies have led to a growing number of high-resolution structures in recent years (Varani and Pardi 1995). Here, we review the application of NMR spectroscopy to RNA structure determination. Practical aspects of a successful NMR study are highlighted, including system design, synthesis of RNAs for NMR, up-to-date methods for spectral analysis and interpretation, and calculation of RNA structures from NMR data. For definitions of the various terms in NMR spectroscopy, the reader is referred to Homans (1992). What NMR can tell us about RNA structure is illustrated by recent NMR studies of RNA.

THEORY
NMR is a spectroscopic method that probes the spin states of nuclei. In the presence of a magnetic field, these spin states have different energies such that transitions between the states can be monitored. Nuclei with spin = 1/2, such as ¹H, ³¹P, ¹⁵N, and ¹³C, have two energy levels in the presence of a magnetic field, which gives the simplest spectroscopic behavior. The latter two isotopes, although not significantly present at natural abundance, can be incorporated into RNAs and proteins at close to 100% abundance using synthetic and biosynthetic methods. The energy-splitting between the two energy states in ¹H NMR is small compared to the splitting between electronic energy levels (UV spectroscopy) and directly proportional to the magnetic field strength; in the presence of an 11.7 Tesla field, the energy-splitting corresponds to a frequency...