Accurate Determination of the RNA Junctions via Single-Molecule High-Precision FRET Measurements

Abstract

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing the biomolecular structure. We apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements (eTCSPC) to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) which are derived from the hairpin ribozyme.Overall 283 FRET pairs were measured with single-molecule MFD and analyzed with the analysis toolkit [1] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.In order to study the influence of the junction on the RNA structure we studied the functional junction part with prolonged helices. Bulge and sequence variations were considered as dominant factors influencing junction conformations for RNA 3WJ. Six different RNA 3WJ with different sequences were studied, two of which have two and five unpaired nucleotides in the junction region.We found three different conformers for RNA 4WJ. However RNAs 3WJ have only one predominant conformer. Furthermore we report that bulges in the junction region determine orientation and rotation of helices, inducing coaxial stacking. Noteworthy the stacked helices are different for the 3WJs with different bulges. Our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality.[1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012).