RNA Junctions Structure and Distance Determination via Accurate Single-Molecule High-Precision FRET Measurements

Abstract

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing biomolecular structures. Here we apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) with a high level of precision in distance better than 1% of the Forster radius [1].We have generated a database of RNA 4WJs and six different RNA 3WJs with different bulges and sequences to study the influence of these factors on the junction conformations of RNA 3WJs. Overall 260 FRET pairs were measured with single-molecule MFD at 20 mM MgCl2 concentration and analyzed with the analysis toolkit [2] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.Monte Carlo simulations showed that sterically allowed conformational space for RNA junctions is large. However, FRET measurements detect the existence of three different static conformers for RNA 4WJ, whereas RNA 3WJs have only one predominant static conformation. terically allowed conformational space for RNA is large. Their junction geometry was described in terms of mutual and Euler angles between helices. The FRET-derived structures suggest that the sequence dictates a junction specific conformation within the large topology space. Furthermore we see that bulges in the junction region determine orientation and rotation of helices and induce coaxial stacking between two of them.[1] Antonik, M., et al., J.Phys.Chem.B, 110, 6970-6978 (2006)[2] Kalinin, S. et al, Nat. Meth., 9, 1218-1225 (2012)