BackgroundSecondary structure forms an important intermediate level of description of nucleic acids that encapsulates the dominating part of the folding energy, is often well conserved in evolution, and is routinely used as a basis to explain experimental findings. Based on carefully measured thermodynamic parameters, exact dynamic programming algorithms can be used to compute ground states, base pairing probabilities, as well as thermodynamic properties.ResultsThe Package has been a widely used compilation of RNA secondary structure related computer programs for nearly two decades. Major changes in the structure of the standard energy model, the Turner 2004 parameters, the pervasive use of multi-core CPUs, and an increasing number of algorithmic variants prompted a major technical overhaul of both the underlying and the interactive user programs. New features include an expanded repertoire of tools to assess RNA-RNA interactions and restricted ensembles of structures, additional output information such as centroid structures and maximum expected accuracy structures derived from base pairing probabilities, or z-scores for locally stable secondary structures, and support for input in format. Updates were implemented without compromising the computational efficiency of the core algorithms and ensuring compatibility with earlier versions.ConclusionsThe , supporting concurrent computations , can be downloaded from http://www.tbi.univie.ac.at/RNA.

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5′ and 3′ mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.

A 3D model of RNA structure can provide information about its function and regulation that is not possible with just the sequence or secondary structure. Current models suffer from low accuracy and long running times and either neglect or presume knowledge of the long-range interactions which stabilize the tertiary structure. Our coarse-grained, helix-based, tertiary structure model operates with only a few degrees of freedom compared with all-atom models while preserving the ability to sample tertiary structures given a secondary structure. It strikes a balance between the precision of an all-atom tertiary structure model and the simplicity and effectiveness of a secondary structure representation. It provides a simplified tool for exploring global arrangements of helices and loops within RNA structures. We provide an example of a novel energy function relying only on the positions of stems and loops. We show that coupling our model to this energy function produces predictions as good as or better than the current state of the art tools. We propose that given the wide range of conformational space that needs to be explored, a coarse-grain approach can explore more conformations in less iterations than an all-atom model coupled to a finegrain energy function. Finally, we emphasize the overarching theme of providing an ensemble of predicted structures, something which our tool excels at, rather than providing a handful of the lowest energy structures.

Motivation: RNA secondary structure contains many non-canonical base pairs of different pair families. Successful prediction of these structural features leads to improved secondary structures with applications in tertiary structure prediction and simultaneous folding and alignment.Results: We present a theoretical model capturing both RNA pair families and extended secondary structure motifs with shared nucleotides using 2-diagrams. We accompany this model with a number of programs for parameter optimization and structure prediction.Availability: All sources (optimization routines, RNA folding, RNA evaluation, extended secondary structure visualization) are published under the GPLv3 and available at www.tbi.univie.ac.at/software/rnawolf/.Contact: choener@tbi.univie.ac.at

RNA has become an integral building material in synthetic biology. Dominated by their secondary structures, which can be computed efficiently, RNA molecules are amenable not only to in vitro and in vivo selection, but also to rational, computation-based design. While the inverse folding problem of constructing an RNA sequence with a prescribed ground-state structure has received considerable attention for nearly two decades, there have been few efforts to design RNAs that can switch between distinct prescribed conformations. We introduce a user-friendly tool for designing RNA sequences that fold into multiple target structures. The underlying algorithm makes use of a combination of graph coloring and heuristic local optimization to find sequences whose energy landscapes are dominated by the prescribed conformations. A flexible interface allows the specification of a wide range of design goals. We demonstrate that bi- and tri-stable "switches" can be designed easily with moderate computational effort for the vast majority of compatible combinations of desired target structures. RNAdesign is freely available under the GPL-v3 license.

Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

G-quadruplexes are abundant locally stable structural elements in nucleic acids. The combinatorial theory of RNA structures and the dynamic programming algorithms for RNA secondary structure prediction are extended here to incorporate G-quadruplexes using a simple but plausible energy model. With preliminary energy parameters, we find that the overwhelming majority of putative quadruplex-forming sequences in the human genome are likely to fold into canonical secondary structures instead. Stable G-quadruplexes are strongly enriched, however, in the 5'UTR of protein coding mRNAs.

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