Precise mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron-Electron Resonance (DEER) Spectroscopy

Dynamic RNA structural changes are critical in biology, and a continuing challenge is to develop methods for precision mapping of these structures. Inter-label distance measurements by DEER (Double-Electron Electron Resonance) spectroscopy in conjunction with side-directed RNA spin labeling provide precise nanoscale structural constraints for biomolecules. This technique has been demonstrated in helical and hairpin RNAs, but has rarely been applied to complex RNA structures. We have used DEER to monitor a large-scale Mg2+-triggered RNA folding transition in the Hammerhead ribozyme, a three-helix junction motif that undergoes an inactive-to-active structural change upon addition of Mg2+. A distinct increase in the population of ribozymes is observed with a short inter-label distance with increasing [Mg2+]. The measured inter-label distance is remarkably consistent with models generated from static crystal structures when it is assumed that the spin labels preferentially localize near to the RNA minor grooves. Data from labels located close to the catalytic core of the ribozyme will probe a putative local structural change in this RNA that may be linked to its catalytic activity. The DEER technique described here can be applied to predict folding of other functional RNA molecules, including those found in complex RNA-protein complexes.1. Kim, N.-K.; Bowman, M.K.; DeRose, V.J. “Precise Mapping of RNA Tertiary Structure via Nanometer Distance Measurements with Double Electron-Electron Resonance Spectroscopy (DEER) Spectroscopy” J. Amer. Chem. Soc. 2010, 132, 8882-8884.