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Ion-mediated electrostatic interactions play an important role in RNA folding stability. For a RNA in a solution with higher Mg2+ ion concentration, more counterions in the solution can bind to the RNA, causing a strong many-body coupling between the bound ions. The many-body effect can change the effective potential of mean force between the tightly bound ions. This effect tends to dampen ion binding and lower RNA folding stability. Neglecting the many-body effect leads to a systematic error (over-estimation) of RNA folding stability at high Mg2+ ion concentrations. Using the tightly bound ion model combined with a conformational ensemble model, we investigate the influence of the many-body effect on the ion-dependent RNA folding stability. Comparisons with the experimental data indicate that including the many-body effect led to much improved predictions for RNA folding stability at high Mg2+ ion concentrations. The results suggest that the many-body effect can be important for RNA folding in high concentrations of multivalent ions. Further investigation showed that the many-body effect can influence the spatial distribution of the tightly bound ions and the effect is more pronounced for compact RNA structures and structures prone to the formation of local clustering of ions. [ABSTRACT FROM AUTHOR]

We develop a polymer physics-based method to compute the conformational entropy for RNA tertiary folds, namely, conformations consisting of multiple helices connected through (cross-linked) loops. The theory is based on a virtual bond conformational model for the nucleotide chain. A key issue in the calculation of the entropy is how to treat the excluded volume interactions. The weak excluded volume interference between the different loops leads to the decomposition of the whole structure into a number of three-body building blocks, each consisting of a loop and two helices connected to the two ends of the loop. The simple construct of the three-body system allows an accurate computation for the conformational entropy for each building block. The assembly of the building blocks gives the entropy of the whole structure. This approach enables treatment of molten globule-like folds (partially unfolded tertiary structures) for RNAs. Extensive tests against experiments and exact computer enumerations indicate that the method can give accurate results for the entropy. The method developed here provides a solid first step toward a systematic development of a theory for the entropy and free energy landscape for complex tertiary folds for RNAs and proteins. [ABSTRACT FROM AUTHOR]

We develop a statistical mechanical framework for the folding thermodynamics of pseudoknotted structures. As applications of the theory, we investigate the folding stability and the free energy landscapes for both the thermal and the mechanical unfolding of pseudoknotted chains. For the mechanical unfolding process, we predict the force-extension curves, from which we can obtain the information about structural transitions in the unfolding process. In general, a pseudoknotted structure unfolds through multiple structural transitions. The interplay between the helix stems and the loops plays an important role in the folding stability of pseudoknots. For instance, variations in loop sizes can lead to the destabilization of some intermediate states and change the (equilibrium) folding pathways (e.g., two helix stems unfold either cooperatively or sequentially). In both thermal and mechanical unfolding, depending on the nucleotide sequence, misfolded intermediate states can emerge in the folding process. In addition, thermal and mechanical unfoldings often have different (equilibrium) pathways. For example, for certain sequences, the misfolded intermediates, which generally have longer tails, can fold, unfold, and refold again in the pulling process, which means that these intermediates can switch between two different average end-end extensions. [ABSTRACT FROM AUTHOR]

The current RNA structure prediction methods cannot keep up the pace of the rapidly increasing number of sequences and the emerging new functions of RNAs. Template-based RNA three-dimensional structure prediction methods are restricted by the limited number of known RNA structures, and traditional motif-based search for the templates does not always lead to successful results. Here we report a new template search and assembly algorithm, the hierarchical loop template-assembly method (VfoldLA). The method searches for templates for single strand loop/junctions instead of the whole motifs, which often renders no available templates, or short fragments (several nucleotides), which requires a long computational time to assemble and refine. The VfoldLA method has the advantage of accounting for local and nonlocal interloop interactions. Benchmark tests indicate that this new method can provide low-resolution predictions for RNA conformations at different levels of structural complexities. Furthermore, the VfoldLA-predicted conformations may also serve as reliable putative models for further structure prediction and refinements. VfoldLA is accessible at http://rna.physics.missouri.edu/vfoldLA. [ABSTRACT FROM AUTHOR]

One of the key issues in the theoretical prediction of RNA folding is the prediction of loop structure from the sequence. RNA loop free energies are dependent on the loop sequence content. However, most current models account only for the loop length-dependence. The previously developed "Vfold" model (a coarse-grained RNA folding model) provides an effective method to generate the complete ensemble of coarse-grained RNA loop and junction conformations. However, due to the lack of sequence-dependent scoring parameters, the method is unable to identify the native and near-native structures from the sequence. In this study, using a previously developed iterative method for extracting the knowledge-based potential parameters from the known structures, we derive a set of dinucleotide-based statistical potentials for RNA loops and junctions. A unique advantage of the approach is its ability to go beyond the the (known) native structures by accounting for the full free energy landscape, including all the nonnative folds. The benchmark tests indicate that for given loop/junction sequences, the statistical potentials enable successful predictions for the coarse-grained 3D structures from the complete conformational ensemble generated by the Vfold model. The predicted coarse-grained structures can provide useful initial folds for further detailed structural refinement. [ABSTRACT FROM AUTHOR]

Transitions between the different conformational states play a critical role in many RNA catalytic and regulatory functions. In this study, we use the Kinetic Monte Carlo method to investigate the kinetic mechanism for the conformational switches between bistable RNA hairpins. We find three types of conformational switch pathways for RNA hairpins: refolding after complete unfolding, folding through basepair-exchange pathways and through pseudoknot-assisted pathways, respectively. The result of the competition between the three types of pathways depends mainly on the location of the rate-limiting base stacks (such as the GC base stacks) in the structures. Depending on the structural relationships between the two bistable hairpins, the conformational switch can follow single or multiple dominant pathways. The predicted folding pathways are supported by the activation energy results derived from the Arrhenius plot as well as the NMR spectroscopy data. [ABSTRACT FROM AUTHOR]

RNA folding is a remarkably complex problem that involves ion-mediated electrostatic interaction, conformational entropy, base pairing and stacking, and noncanonical interactions. During the past decade, results from a variety of experimental and theoretical studies pointed to (a) the potential ion correlation effect in Mg2+-RNA interactions, (b) the rugged energy landscapes and multistate RNA folding kinetics even for small RNA systems such as hairpins and pseudoknots, (c) the infraloop interactions and sequence-dependent loop free energy, and (d) the strong nonadditivity of chain entropy in RNA pseudoknot and other tertiary folds. Several related issues, which have not been thoroughly resolved, require combined approaches with thermodynamic and kinetic experiments, statistical mechanical modeling, and all-atom computer simulations. [ABSTRACT FROM AUTHOR]

Examines the RNA hairpin-folding kinetics. Creation of statistical mechanics model to combine eigenvalue solution; Visualization of distinct time separation and rate of limiting step; Involvement of trapping and detrapping in folding of kinetic intermediates.

Terahertz time-domain spectroscopy has been employed to measure the optical constants of ytterbium-doped yttrium aluminum garnet (YbxY1-x)3Al5O12 (Yb:YAG) crystals for nominal x values of 0.0, 0.1, 0.2, 0.5, 0.8, and 1.0 in the frequency range from 0.2 to 1.8 THz. The real refractive indices for Yb:YAG crystals increase linearly with the concentration of Yb3+. The experimental results can be fitted by the Sellmeier equation. The results imply that the phonon modes of the Yb:YAG crystals shift to lower frequencies with substitution of Yb for Y. The extinction coefficients of the Yb:YAG crystals are smaller than 0.05. [ABSTRACT FROM AUTHOR]

RNA functions are intrinsically tied tofolding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/ dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model. [ABSTRACT FROM AUTHOR]

Background: In this study, we sequenced and phylogenetic analyses of the VP2 genes from twelve canine parvovirus (CPV) strains obtained from eleven domestic dogs and a giant panda (Ailuropoda melanoleuca) in China. A novel canine parvovirus (CPV) was detected from the giant panda in China. Results: Nucleotide and phylogenetic analysis of the capsid protein VP2 gene classified the CPV as a new CPV-2a type. Substitution of Gln for Arg at the conserved 370 residue in CPV presents an unusual variation in the new CPV-2a amino acid sequence of the giant panda and is further evidence for the continuing evolution of the virus. Conclusions: These findings extend the knowledge on CPV molecular epidemiology of particular relevance to wild carnivores. [ABSTRACT FROM AUTHOR]

Objective To investigate the clinical characteristics, operative methods, and diffusion tensor imaging (DTI) in the resection of intrinsic insular gliomas via transsylvian approach. Methods From June 2008 to June 2010, 12 patients with intrinsic insular gliomas were treated via transsylvian microsurgical approach, with preoperative magnetic resonance imaging diffusion tensor imaging (MR DTI) evaluation. The data of these patients were retrospectively analyzed. Results All patients had astrocytoma, including 8 patients of Grades I to II, 2 patients of Grades III to IV, and 2 patients of mixed glial tumors. The insular tumors were completely removed in 9 patients, whereas they were only partially removed from 3 patients. No death was related to the operations. Two patients had transient aphasia, 2 experienced worsened hemiplegia on opposite sides of their bodies, and 2 had mild hemiplegia and language function disturbance. Conclusions Most of the insular gliomas are of low grade. By evaluating the damage of the corticospinal tract through DTI and using ultrasonography to locate the tumors during operation, microsurgery treatment removes the lesions as much as possible, protects the surrounding areas, reduces the mobility rate, and improves the postoperative quality of life. [ABSTRACT FROM AUTHOR]

While CRISPR/Cas9 is a powerful tool in genome engineering, the ontarget activity and off-target effects of the system widely vary because of the differences in guide RNA (gRNA) sequences and genomic environments. Traditional approaches rely on separate models and parameters to treat on- and off-target cleavage activities. Here, we demonstrate that a free-energy scheme dominates the Cas9 editing efficacy and delineate a method that simultaneously considers on-target activities and off-target effects. While data-driven machinelearning approaches learn rules to model particular datasets, they may not be as transferrable to new systems or capable of producing new mechanistic insights as principled physical approaches. By integrating the energetics of R-loop formation under Cas9 binding, the effect of the protospacer adjacent motif sequence, and the folding stability of the whole single guide RNA, we devised a unified, physical model that can apply to any cleavage-activity dataset. This unified framework improves predictions for both on-target activities and offtarget efficiencies of spCas9 and may be readily transferred to other systems with different guide RNAs or Cas9 ortholog proteins. [ABSTRACT FROM AUTHOR]

The selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method probes RNA local structural and dynamic information at single nucleotide resolution. To gain quantitative insights into the relationship between nucleotide flexibility, RNA 3D structure, and SHAPE reactivity, we develop a 3D Structure-SHAPE Relationship model (3DSSR) to rebuild SHAPE profiles from 3D structures. The model starts from RNA structures and combines nucleotide interaction strength and conformational propensity, ligand (SHAPE reagent) accessibility, and base-pairing pattern through a composite function to quantify the correlation between SHAPE reactivity and nucleotide conformational stability. The 3DSSR model shows the relationship between SHAPE reactivity and RNA structure and energetics. Comparisons between the 3DSSR-predicted SHAPE profile and the experimental SHAPE data show correlation, suggesting that the extracted analytical function may have captured the key factors that determine the SHAPE reactivity profile. Furthermore, the theory offers an effective method to sieve RNA 3D models and exclude models that are incompatible with experimental SHAPE data. [ABSTRACT FROM AUTHOR]

We develop a partial charge-based tightly bound ion (PCTBI) model for the ion effects in RNA folding. On the basis of the Monte Carlo tightly bound ion (MCTBI) approach, the model can account for ion fluctuation and correlation effects, and can predict the ion distribution around the RNA. Furthermore, unlike the previous coarse-grained RNA charge models, where negative charges are placed on the phosphates only, the current new model considers the detailed all-atom partial charge distribution on the RNA. Thus, the model not only keeps the advantage of the MCTBI model, but also has the potential to provide important detailed information unattainable by the previous MCTBI models. For example, the model predicts the reduction in ion binding upon protein binding and ion-induced conformational switches. For hepatitis C virus genomic RNA, the model predicts a Mg2+-induced stabilization of a kissing motif for a cis-acting regulatory element in the genomic RNA. Extensive theory-experiment comparisons support the reliability of the theoretical predictions. Therefore, the model may serve as a robust starting point for further development of an accurate method for ion effects in an RNA conformational equilibrium and RNA-cofactor interactions. [ABSTRACT FROM AUTHOR]

We develop a new statistical mechanical model to predict the molecular crowding effects in ion-RNA interactions. By considering discrete distributions of the crowders, the model can treat the main crowder-induced effects, such as the competition with ions for RNA binding, changes of electrostatic interaction due to crowder-induced changes in the dielectric environment, and changes in the nonpolar hydration state of the crowder-RNA system. To enhance the computational efficiency, we sample the crowder distribution using a hybrid approach: For crowders in the close vicinity of RNA surface, we sample their discrete distributions; for crowders in the bulk solvent away from the RNA surface, we use a continuous mean-field distribution for the crowders. Moreover, using the tightly bound ion (TBI) model, we account for ion fluctuation and correlation effects in the calculation for ion-RNA interactions. Applications of the model to a variety of simple RNA structures such as RNA helices show a crowder-induced increase in free energy and decrease in ion binding. Such crowding effects tend to contribute to the destabilization of RNA structure. Further analysis indicates that these effects are associated with the crowder-ion competition in RNA binding and the effective decrease in the dielectric constant. This simple ion effect model may serve as a useful framework for modeling more realistic crowders with larger, more complex RNA structures. [ABSTRACT FROM AUTHOR]

Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot's unfolding s intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5' to 3' end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5' to 3' end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5' or 3' ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5' → 3' direction and Helix-2 in the 3' → 5' direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including ffameshifting. [ABSTRACT FROM AUTHOR]

Background: Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China. Results: Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species. Conclusions: These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-cainds in China. [ABSTRACT FROM AUTHOR]

Transitions between different conformational states, so-called conformational switching, are intrinsic to RNA catalytic and regulatory functions. Often, conformational switching occurs on time scales of several seconds. In combination with the recent real-time NMR experiments (Wenter et al. Angew. Chem. Int. Ed.2005, 44, 2600; Wenter et al. ChemBioChem2006, 7, 417) for the transitions between bistable RNA conformations, we combine the master equation method with the kinetic cluster method to investigate the detailed kinetic mechanism and the factors that govern the folding kinetics. We propose that heat capacity change (ΔCp) upon RNA folding may be important for RNA folding kinetics. In addition, we find that, for tetraloop hairpins, noncanonical (tertiary) intraloop interactions are important to determine the folding kinetics. Furthermore, through theory−experiment comparisons, we find that the different rate models for the fundamental steps (i.e., formation/disruption of a base pair or stack) can cause contrasting results in the theoretical predictions. [ABSTRACT FROM AUTHOR]