Ctures for each construct, the lowest average RMSD values were 4.2 for LCS1co and 3.3 for LCS2co; the probabilities of obtaining these RMSD values in the structure ensembles are 0.085 and 0.007, respectively, indicating the greater significance of the predicted LCS2co structure. With a more stringent criterion combining the averagebinding energies of the 3D models in different ionic conditions to gain insight into how environmental factors impact miRNA arget recognition. Finally, we explored how different structural features of duplexes influence the stability of Argonaute NA interactions by performing docking simulations.3D-based algorithm reproduces NMR structures of let-7 arget constructs We first assessed performance by comparing our 3D predictions with solution structures of miRNA arget duplexes obtained by NMR spectroscopy. The C. elegans let-7 miRNA is known to bind two complementary sites in the lin-41 3 UTR that are separated by 27 nt (Vella et al. 2004). Both let-7 complementary sites (LCSs) are required for down-regulation of lin-41, although the mechanism of their cooperative function is not yet understood (Vella et al.Losmapimod 2004).Ethotoin Recently, solution structures have been solved for let-7-LCS duplexes from both sites (LCS1co and LCS2co) using single-chain constructs with a closing loop and modified bases to enhanceFIGURE 2. NMR models and predicted structures for single-stranded constructs of C. elegans let-7 miRNA arget site duplexes (A) LCS1co and (B) LCS2co. (Left) Superposition of 10 lowest-energy NMR structures for each construct. (Middle) Structure alignments of individual representative NMR (wheat color) and predicted (light blue) structures, highlighting bases in the internal loop (NMR, red; predicted, blue).PMID:24883330 (Right) Magnified view of internal loops from aligned structures, with bases labeled. For substructure alignments, structures were partitioned into internal loop, hairpin (upper), and stem (lower) regions. The predicted structures shown have average RMSD values of 4.2 for LCS1co and 3.3 for LCS2co (relative to the 10 lowest-energy NMR structures for the corresponding construct).www.rnajournal.orgGan and GunsalusTABLE 1. RMSD values ( for NMR structures, best structures found in our structure ensembles, and predicted structures from this work and FARNA NMR LCS1co LCS2co 1.9 1.2 Best RMSD 2.8 3.0 This work 4.2 3.3 FARNA 4.7 4.RMSDs of all top five structures, the mean RMSD was 5.2 for LCS1co and 4.2 for LCS2co. The lowest average RMSD values in the ensembles were 2.8 and 3.0 but these structures had slightly higher energies than the top-ranking structures because the model energy function used is imperfect. The better predictive performance we see (smaller RMSD) for LCS2co could reflect its higher stability, as observed experimentally (the melting temperature of LCS2co is 7 K higher than that of LCS1co) (Cevec et al. 2008, 2010). Overall, these results indicate that our structure assembly procedures sampled adequately small RMSD structures (e.g., the ensemble sizes required to attain the lowest RMSD values were well below the 1000: 281 for LCS1co and 21 for LCS2co). The RMSD values obtained here are comparable to those from previous studies of similarly sized RNAs; for example, the MC-Sym method reported an average RMSD of 2.7 for best structures of 30- to 47-nt RNAs (Parisien and Major 2008), and the FARNA approach yielded an average RMSD of 3.4 for 28- to 46-nt RNAs (Das and Baker 2007). This comparison of our best p.