Combining temperature and force to study folding of an RNA hairpin.

RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.

MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression.

MicroRNAs (miRNAs) are a class of short ( approximately 22-nt) noncoding RNA molecules that downregulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in Caenorhabditis elegans, hundreds of miRNAs have been identified in both animals and plants. Here, we report a technique for visualizing detailed miRNA expression patterns in mouse embryos. We elucidate the tissue-specific expression of several miRNAs during embryogenesis, including two encoded by genes embedded in homeobox (Hox) clusters, miR-10a and miR-196a. These two miRNAs are expressed in patterns that are markedly reminiscent of those of Hox genes. Furthermore, miR-196a negatively regulates Hoxb8, indicating that its restricted expression pattern probably reflects a role in the patterning function of the Hox complex.

Comparing RNA secondary structures using a relaxed base-pair score.

The use of free energy-based algorithms to compute RNA secondary structures produces, in general, large numbers of foldings. Recent research has addressed the problem of grouping structures into a small number of clusters and computing a representative folding for each cluster. At the heart of this problem is the need to compute a quantity that measures the difference between pairs of foldings. We introduce a new concept, the relaxed base-pair (RBP) score, designed to give a more biologically realistic measure of the difference between structures than the base-pair (BP) metric, which simply counts the number of base pairs in one structure but not the other. The degree of relaxation is determined by a single relaxation parameter, t. When t = 0, (no relaxation) our method is the same as the BP metric. At the other extreme, a very large value of t will give a distance of 0 for identical structures and 1 for structures that differ. Scores can be recomputed with different values of t, at virtually no extra computation cost, to yield satisfactory results. Our results indicate that relaxed measures give more stable and more meaningful clusters than the BP metric. We also use the RBP score to compute representative foldings for each cluster.

Quantitative prediction of miRNA-mRNA interaction based on equilibrium concentrations.

MicroRNAs (miRNAs) suppress gene expression by forming a duplex with a target messenger RNA (mRNA), blocking translation or initiating cleavage. Computational approaches have proven valuable for predicting which mRNAs can be targeted by a given miRNA, but currently available prediction methods do not address the extent of duplex formation under physiological conditions. Some miRNAs can at low concentrations bind to target mRNAs, whereas others are unlikely to bind within a physiologically relevant concentration range. Here we present a novel approach in which we find potential target sites on mRNA that minimize the calculated free energy of duplex formation, compute the free energy change involved in unfolding these sites, and use these energies to estimate the extent of duplex formation at specified initial concentrations of both species. We compare our predictions to experimentally confirmed miRNA-mRNA interactions (and non-interactions) in Drosophila melanogaster and in human. Although our method does not predict whether the targeted mRNA is degraded and/or its translation to protein inhibited, our quantitative estimates generally track experimentally supported results, indicating that this approach can be used to predict whether an interaction occurs at specified concentrations. Our approach offers a more-quantitative understanding of post-translational regulation in different cell types, tissues, and developmental conditions.

Development of lead hammerhead ribozyme candidates against human rod opsin mRNA for retinal degeneration therapy.

To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half-life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p<0.05). Successful lead candidates (hhRz CUC [see in text downward arrow] 266, hhRz CUC [see in text downward arrow] 1411, hhRz AUA [see in text downward arrow] 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48h paradigm of testing. These results validate a rigorous computational bioinformatics approach to detect accessible regions of target mRNAs in cellulo. The opsin knockdown effect could prove to be clinically significant when integrated over longer periods in photoreceptors. Further optimization and animal testing are the next step in this stratified RNA drug discovery program. A recently developed novel and efficient screening assay based upon expression of a dicistronic mRNA (RHO-IRES-SEAP) containing both RHO and reporter (SEAP) cDNAs was used to compare the hhRz 266 lead candidate to another agent (Rz525/hhRz485) already known to partially rescue retinal degeneration in a rodent model. Lead hhRz 266 CUC [see in text downward arrow] proved more efficacious than Rz525/hhRz485 which infers viability for rescue of retinal degeneration in appropriate preclinical models of disease.

Morphological, molecular, and phylogenetic characterization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera.

Nosema ceranae, a microsporidian parasite originally described from Apis cerana, has been found to infect Apis melllifera and is highly pathogenic to its new host. In the present study, data on the ultrastructure of N. ceranae, presence of N. ceranae-specific nucleic acid in host tissues, and phylogenetic relationships with other microsporidia species are described. The ultrastructural features indicate that N. ceranae possesses all of the characteristics of the genus Nosema. Spores of N. ceranae measured approximately 4.4 x 2.2 µm on fresh smears. The number of coils of the polar filament inside spores was 18-21. Polymerase chain reaction (PCR) signals specific for N. ceranae were detected not only in the primary infection site, the midgut, but also in the tissues of hypopharyngeal glands, salivary glands, Malpighian tubules, and fat body. The detection rate and intensity of PCR signals in the fat body were relatively low compared with other examined tissues. Maximum parsimony analysis of the small subunit rRNA gene sequences showed that N. ceranae appeared to be more closely related to the wasp parasite, Nosema vespula, than to N. apis, a parasite infecting the same host.

UNAFold: software for nucleic acid folding and hybridization.

The UNAFold software package is an integrated collection of programs that simulate folding, hybridization, and melting pathways for one or two single-stranded nucleic acid sequences. The name is derived from "Unified Nucleic Acid Folding." Folding (secondary structure) prediction for single-stranded RNA or DNA combines free energy minimization, partition function calculations and stochastic sampling. For melting simulations, the package computes entire melting profiles, not just melting temperatures. UV absorbance at 260 nm, heat capacity change (C(p)), and mole fractions of different molecular species are computed as a function of temperature. The package installs and runs on all Unix and Linux platforms that we have looked at, including Mac OS X. Images of secondary structures, hybridizations, and dot plots may be computed using common formats. Similarly, a variety of melting profile plots is created when appropriate. These latter plots include experimental results if they are provided. The package is "command line" driven. Underlying compiled programs may be used individually, or in special combinations through the use of a variety of Perl scripts. Users are encouraged to create their own scripts to supplement what comes with the package. This evolving software is available for download at http://www.bioinfo.rpi.edu/applications/hybrid/download.php .

DINAMelt web server for nucleic acid melting prediction.

The DINAMelt web server simulates the melting of one or two single-stranded nucleic acids in solution. The goal is to predict not just a melting temperature for a hybridized pair of nucleic acids, but entire equilibrium melting profiles as a function of temperature. The two molecules are not required to be complementary, nor must the two strand concentrations be equal. Competition among different molecular species is automatically taken into account. Calculations consider not only the heterodimer, but also the two possible homodimers, as well as the folding of each single-stranded molecule. For each of these five molecular species, free energies are computed by summing Boltzmann factors over every possible hybridized or folded state. For temperatures within a user-specified range, calculations predict species mole fractions together with the free energy, enthalpy, entropy and heat capacity of the ensemble. Ultraviolet (UV) absorbance at 260 nm is simulated using published extinction coefficients and computed base pair probabilities. All results are available as text files and plots are provided for species concentrations, heat capacity and UV absorbance versus temperature. This server is connected to an active research program and should evolve as new theory and software are developed. The server URL is http://www.bioinfo.rpi.edu/applications/hybrid/.

Mfold web server for nucleic acid folding and hybridization prediction.

The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as 'MFOLDROOT'.

OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach.

There is a substantial interest in implementing bioinformatics technologies that allow the design of oligonucleotides to support the development of microarrays made from short synthetic DNA fragments spotted or in situ synthesized on slides. Ideally, such oligonucleotides should be totally specific to their respective targets to avoid any cross-hybridization and should not form stable secondary structures that may interfere with the labeled probes during hybridization. We have developed OligoArray 2.0, a program that designs specific oligonucleotides at the genomic scale. It uses a thermodynamic approach to predict secondary structures and to calculate the specificity of targets on chips for a unique probe in a mixture of labeled probes. Furthermore, OligoArray 2.0 can adjust the oligonucleotide length, according to user input, to fit a narrow T(m) range compatible with hybridization requirements. Combined with on chip oligonucleotide synthesis, this program makes it feasible to perform expression analysis on a genomic scale for any organism for which the genome sequence is known. This is without relying on cDNA or oligonucleotide libraries. OligoArray 2.0 was used to design 75 764 oligonucleotides representing 26 140 transcripts from Arabidopsis thaliana. Among this set, we provide at least one specific oligonucleotide for 93% of these transcripts.

Transcriptome-wide prediction of miRNA targets in human and mouse using FASTH.

Transcriptional regulation by microRNAs (miRNAs) involves complementary base-pairing at target sites on mRNAs, yielding complex secondary structures. Here we introduce an efficient computational approach and software (FASTH) for genome-scale prediction of miRNA target sites based on minimizing the free energy of duplex structure. We apply our approach to identify miRNA target sites in the human and mouse transcriptomes. Our results show that short sequence motifs in the 5' end of miRNAs frequently match mRNAs perfectly, not only at validated target sites but additionally at many other, energetically favourable sites. High-quality matching regions are abundant and occur at similar frequencies in all mRNA regions, not only the 3'UTR. About one-third of potential miRNA target sites are reassigned to different mRNA regions, or gained or lost altogether, among different transcript isoforms from the same gene. Many potential miRNA target sites predicted in human are not found in mouse, and vice-versa, but among those that do occur in orthologous human and mouse mRNAs most are situated in corresponding mRNA regions, i.e. these sites are themselves orthologous. Using a luciferase assay in HEK293 cells, we validate four of six predicted miRNA-mRNA interactions, with the mRNA level reduced by an average of 73%. We demonstrate that a thermodynamically based computational approach to prediction of miRNA binding sites on mRNAs can be scaled to analyse complete mammalian transcriptome datasets. These results confirm and extend the scope of miRNA-mediated species- and transcript-specific regulation in different cell types, tissues and developmental conditions.

RNA secondary structure prediction.

Details for predicting secondary structure of RNA sequences using free energy minimization are given. Protocols present the use of the RNAstructure computer program (for PCs) and the mfold server (for Unix platforms). The minimum free energy structure and a set of suboptimal structures with similar free energies are predicted. Prediction of high-affinity oligonucleotide binding sites to a structured RNA target is also presented.

Prediction of hybridization and melting for double-stranded nucleic acids.

This article presents a general statistical mechanical approach to describe self-folding together with the hybridization between a pair of finite length DNA or RNA molecules. The model takes into account the entire ensemble of single- and double-stranded species in solution and their mole fractions at different temperatures. The folding and hybridization models deal with matched pairs, mismatches, symmetric and asymmetric interior loops, bulges, and single-base stacking that might exist at duplex ends or at the ends of helices. All possible conformations of the single- and double-stranded species are explored. Only intermolecular basepairs are considered in duplexes at this stage.In particular we focus on the role of stacking between neighboring nucleotide residues of single unfolded strands as an important source of enthalpy change on helix formation which has not been modeled computationally thus far. Changes in the states of the single strands with temperature are shown to lead to a larger heat effect at higher temperature. An important consequence of this is that predictions of enthalpies, which are based on databases of nearest-neighbor energy parameters determined for molecules or duplexes with lower melting temperatures compared with the melting temperatures of the oligos for which they are used as a predictive tool, will be underestimated.

OligoArray: genome-scale oligonucleotide design for microarrays.

SUMMARY: OligoArray is a program that computes gene specific and secondary structure free oligonucleotides for genome-scale oligonucleotide microarray construction or other applications. AVAILABILITY: The program code is distributed under the GNU General Public License and is freely available for non-profit use via request from the authors.

Heterozygous mutation in 5'-untranslated region of sepiapterin reductase gene (SPR) in a patient with dopa-responsive dystonia.

The search for mutations in genes coding for components of the biopterin pathway other than GTPCH1 revealed a mutation in the gene coding for sepiapterin reductase (SPR) in 1 of 95 patients with GCH1-negative dopa-responsive dystonia (DRD). The mutation detected in SPR is a G-->A transition at position -13 of the untranslated region of the gene. This resulted in drastically reduced activity of sepiapterin reductase in the patient's fibroblasts. The findings indicate that haploinsufficiency of SPR can be a rare cause of DRD.

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.

A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

RNAML: a standard syntax for exchanging RNA information.

Analyzing a single data set using multiple RNA informatics programs often requires a file format conversion between each pair of programs, significantly hampering productivity. To facilitate the interoperation of these programs, we propose a syntax to exchange basic RNA molecular information. This RNAML syntax allows for the storage and the exchange of information about RNA sequence and secondary and tertiary structures. The syntax permits the description of higher level information about the data including, but not restricted to, base pairs, base triples, and pseudoknots. A class-oriented approach allows us to represent data common to a given set of RNA molecules, such as a sequence alignment and a consensus secondary structure. Documentation about experiments and computations, as well as references to journals and external databases, are included in the syntax. The chief challenge in creating such a syntax was to determine the appropriate scope of usage and to ensure extensibility as new needs will arise. The syntax complies with the eXtensible Markup Language (XML) recommendations, a widely accepted standard for syntax specifications. In addition to the various generic packages that exist to read and interpret XML formats, an XML processor was developed and put in the open-source MC-Core library for nucleic acid and protein structure computer manipulation.