An example of a pseudo-energy calculation using the NCM model.

<p>ΔG_junction is evaluated for each pair of NCMs that share an edge, i.e. the ones that have an overlapping base pair. This term depends on the identities of the two NCMs (that is, the length of the 5’ and 3’ cycles for a double-stranded NCM, or the total length for a single-stranded NCM), and the nucleotides in the common base pair. This term is evaluated for the junction of NCM a with NCM b, NCM b with NCM c, and NCM c with NCM d.</p

Benchmark of single-sequence prediction of non-canonical base pairs.

<p>(A) Prediction accuracy of the lowest free energy structure, evaluated on non-canonical pairs. This includes a calculation where CycleFold is constrained to include the known canonical base pairs to illustrate the performance of the NCM approach when canonical base pairs are known. (B) Prediction with CycleFold, using structures composed of highly probable non-canonical pairs. Sensitivity and PPV are reported for structures with probability greater than a specified threshold (labeled on the plot). This demonstrates that the threshold stringency provides a tradeoff in terms of sensitivity and PPV.</p

Base pair probability estimates improve the prediction accuracy of RNA non-canonical base pairs - Fig 4

<p>Prediction of canonical base pairs by predicting a conserved structure using multiple homologous sequences for (A) an MVE virus nuclease resistant RNA [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005827#pcbi.1005827.ref053" target="_blank">53</a>], (B) a <i>D</i>. <i>radiodurans</i> SRP hairpin domain [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005827#pcbi.1005827.ref054" target="_blank">54</a>], and (C) a <i>O</i>. <i>sativa</i> Twister ribozyme [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005827#pcbi.1005827.ref055" target="_blank">55</a>]. Prediction accuracy is shown for structures composed of highly probable pairs using information from a single sequence (blue) or a TurboFold calculation with 10 sequences (red). Also shown is prediction accuracy using evolutionary couplings from the plmc program [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005827#pcbi.1005827.ref016" target="_blank">16</a>] (green).</p

A recursion diagram [41] illustrating the NCM partition function algorithm.

<p>Filled regions indicate terms that are being added to the partition function, and empty regions indicate results that were previously calculated. Solid lines indicate nucleotides that must be paired, while dotted lines indicate nucleotides that may or may not be paired.</p

Prediction of extended secondary structure with CycleFold.

<p>(A) A predicted structure for a Sarcin-Ricin loop sequence form <i>Rattus norvegicus</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005827#pcbi.1005827.ref050" target="_blank">50</a>] using CycleFold with the MFE algorithm. Correctly predicted canonical pairs are drawn with heavy black lines, correctly predicted non-canonical pairs are light black lines, and the incorrectly predicted non-canonical pair is shown with a gray dashed line. The G-A pair at the base of the tetraloop is not present in the reference structure because the 3’ A is not stacked on the subsequent G, but is instead in contact with a protein, Restrictocin. (B) The probability dot plot calculated using Cyclefold with the partition function algorithm. The upper right triangle shows pairs with estimated probabilities > 0.01, color-coded by pairing probability. The lower left triangle shows the pairs that are present in the reference structure. Each dot represents a single base pair, and nucleotide index (starting with 1 at the 5’ end) is shown along the x and y axes.</p

The Amber ff99 Force Field Predicts Relative Free Energy Changes for RNA Helix Formation

The ability of the Amber ff99 force field to predict relative free energies of RNA helix formation was investigated. The test systems were three hexaloop RNA hairpins with identical loops and varying stems. The potential of mean force of stretching the hairpins from the native state to an extended conformation was calculated with umbrella sampling. Because the hairpins have identical loop sequence, the differences in free energy changes are only from the stem composition. The Amber ff99 force field was able to correctly predict the order of stabilities of the hairpins, although the magnitude of the free energy change is larger than that determined by optical melting experiments. The two measurements cannot be compared directly because the unfolded state in the optical melting experiments is a random coil, while the end state in the umbrella sampling simulations was an elongated chain. The calculations can be compared to reference data by using a thermodynamic cycle. By applying the thermodynamic cycle to the transitions between the hairpins using simulations and nearest-neighbor data, agreement was found to be within the sampling error of simulations, thus demonstrating that ff99 force field is able to accurately predict relative free energies of RNA helix formation

Fluorescence Competition and Optical Melting Measurements of RNA Three-Way Multibranch Loops Provide a Revised Model for Thermodynamic Parameters

Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure

Clausola sulla decadenza

Lo scritto esamina il contenuto e gli effetti giuridici del patto sulla decadenza.The paper examines the content and legal effects of the patto sulla decadenza

Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics

Modified nucleotides are prevalent in tRNA. Experimental studies reveal that these covalent modifications play an important role in tuning tRNA function. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA dynamics. The X-ray crystal structures of tRNA­(Asp), tRNA­(Phe), and tRNA­(iMet), both with and without modifications, were used as initial structures for 333 ns explicit solvent MD simulations with AMBER. For each tRNA molecule, three independent trajectory calculations were performed, giving an aggregate of 6 μs of total MD across six molecules. The global root-mean-square deviations (RMSD) of atomic positions show that modifications only introduce significant rigidity to the global structure of tRNA­(Phe). Interestingly, RMSDs of the anticodon stem-loop (ASL) suggest that modified tRNA has a more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that the rigidity of the anticodon stem-loop is finely tuned by modifications, where rigidity in the anticodon arm is essential for tRNA translocation in the ribosome, and flexibility of the anticodon is important for codon recognition. Sugar pucker and water residence time of pseudouridines in modified tRNAs and corresponding uridines in unmodified tRNAs were assessed, and the results reinforce that pseudouridine favors the 3′-endo conformation and has a higher tendency to interact with water. Principal component analysis (PCA) was used to examine correlated motions in tRNA. Additionally, covariance overlaps of PCAs were compared for trajectories of the same molecule and between trajectories of modified and unmodified tRNAs. The comparison suggests that modifications alter the correlated motions. For the anticodon bases, the extent of stacking was compared between modified and unmodified molecules, and only unmodified tRNA­(Asp) has significantly higher percentage of stacking time. Overall, the simulations reveal that the effect of covalent modification on tRNA dynamics is not simple, with modifications increasing flexibility in some regions of the structure and increasing rigidity in other regions

Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics

Modified nucleotides are prevalent in tRNA. Experimental studies reveal that these covalent modifications play an important role in tuning tRNA function. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA dynamics. The X-ray crystal structures of tRNA­(Asp), tRNA­(Phe), and tRNA­(iMet), both with and without modifications, were used as initial structures for 333 ns explicit solvent MD simulations with AMBER. For each tRNA molecule, three independent trajectory calculations were performed, giving an aggregate of 6 μs of total MD across six molecules. The global root-mean-square deviations (RMSD) of atomic positions show that modifications only introduce significant rigidity to the global structure of tRNA­(Phe). Interestingly, RMSDs of the anticodon stem-loop (ASL) suggest that modified tRNA has a more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that the rigidity of the anticodon stem-loop is finely tuned by modifications, where rigidity in the anticodon arm is essential for tRNA translocation in the ribosome, and flexibility of the anticodon is important for codon recognition. Sugar pucker and water residence time of pseudouridines in modified tRNAs and corresponding uridines in unmodified tRNAs were assessed, and the results reinforce that pseudouridine favors the 3′-endo conformation and has a higher tendency to interact with water. Principal component analysis (PCA) was used to examine correlated motions in tRNA. Additionally, covariance overlaps of PCAs were compared for trajectories of the same molecule and between trajectories of modified and unmodified tRNAs. The comparison suggests that modifications alter the correlated motions. For the anticodon bases, the extent of stacking was compared between modified and unmodified molecules, and only unmodified tRNA­(Asp) has significantly higher percentage of stacking time. Overall, the simulations reveal that the effect of covalent modification on tRNA dynamics is not simple, with modifications increasing flexibility in some regions of the structure and increasing rigidity in other regions