Accurate Distance and Structure Determination of Three Different RNA Three-Way Junctions via High Precision FRET

RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of such stacking in case with no bulge. noteworthy the stacked helices are different for the 3WJs with two and five nucleotides in the bulge.Our studies showed that high precision FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers.References1. Sindbert S, et al. (2011) Accur ate distance determination of nucleic acids via Forster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133(8):2463-2480.2. Kalinin S,et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods in revision.