Dynalign: an algorithm for finding the secondary structure common to two RNA sequences

With the rapid increase in the size of the genome sequence database, computational analysis of RNA will become increasingly important in revealing structure-function relationships and potential drug targets. RNA secondary structure prediction for a single sequence is 73 % accurate on average for a large database of known secondary structures. This level of accuracy provides a good starting point for determining a secondary structure either by comparative sequence analysis or by the interpretation of experimental studies. Dynalign is a new computer algorithm that improves the accuracy of structure prediction by combining free energy minimization and comparative sequence analysis to find a low free energy structure common to two sequences without requiring any sequence identity. It uses a dynamic programming construct suggested by Sankoff. Dynalign, however, restricts the maximum distance, M, allowed between aligned nucleotides in the two sequences. This makes the calculation tractable because the complexity is simplified to O(M(3)N(3)), where N is the length of the shorter sequence.The accuracy of Dynalign was tested with sets of 13 tRNAs, seven 5 S rRNAs, and two R2 3' UTR sequences. On average, Dynalign predicted 86.1 % of known base-pairs in the tRNAs, as compared to 59.7 % for free energy minimization alone. For the 5 S rRNAs, the average accuracy improves from 47.8 % to 86.4 %. The secondary structure of the R2 3' UTR from Drosophila takahashii is poorly predicted by standard free energy minimization. With Dynalign, however, the structure predicted in tandem with the sequence from Drosophila melanogaster nearly matches the structure determined by comparative sequence analysis.     

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.     

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure

Binding of small oligonucleotides to the periphery of folded RNA can provide insight into the secondary structure of complex RNA in solution. To discriminate between bound and unbound fluorescein-labeled 2'-O-methyl RNA probes, we use ionically coated gold nanoparticles to selectively adsorb unbound probes and quench their fluorescence. The target is the 3' untranslated region of Bombyx mori R2 RNA. Fluorescence indicates that R2 sequences complementary to some of the probes are accessible for binding in the three-dimensional structure. Hybridization occurs under homogeneous conditions in the absence of the gold nanoparticles so that steric issues associated with chip-based assays are avoided. The assay is compatible with well plate formats, takes less than 5 min, and requires only 2 pmol or less of unlabeled target RNA per probe sequence tested.     

Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3' end of Bombyx mori R2 RNA

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.     

Structural analysis of a group II intron by chemical modifications and minimal energy calculations.

Folding of the yeast mitochondrial group II intron aI5c has been analysed by chemical modification of the in vitro synthesised RNA with dimethylsulfate and diethylpyrocarbonate. Computer calculations of the intron secondary structure through minimization of free energy were also performed in order to study thermodynamic properties of the intron and to relate these to data obtained from chemical modification. Comparison of the two sets of data with the current phylogenetic model structure of the intron aI5 reveals close agreement, thus lending strong support for the existence of a typical group II intron core structure comprising six neighbouring stem-loop domains. Local discrepancies between the experimental data and the model structures have been analyzed by reference to thermodynamic properties of the structure. This shows that use of the latest refined set of free energy values improves the structure calculation significantly.     

Secondary Structures for 5′ Regions of R2 Retrotransposon RNAs Reveal a Novel Conserved Pseudoknot and Regions that Evolve under Different Constraints

Sequences from the 5′ region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for a segment of this RNA is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five conserved secondary structures are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5′ segment to a protein coding function near the 3′ end. This suggests that local regions evolved under separate functional constraints (structural, coding, or both).     

Thermodynamic characterization of the complete set of sequence symmetric tandem mismatches in RNA and an improved model for predicting the free energy contribution of sequence asymmetric tandem mismatches

Because of the availability of an abundance of RNA sequence information, the ability to rapidly and accurately predict the secondary structure of RNA from sequence is becoming increasingly important. A common method for predicting RNA secondary structure from sequence is free energy minimization. Therefore, accurate free energy contributions for every RNA secondary structure motif are necessary for accurate secondary structure predictions. Tandem mismatches are prevalent in naturally occurring sequences and are biologically important. A common method for predicting the stability of a sequence asymmetric tandem mismatch relies on the stabilities of the two corresponding sequence symmetric tandem mismatches [Mathews, D. H., Sabina, J., Zuker, M., and Turner, D. H. (1999) J. Mol. Biol. 288, 911-940]. To improve the prediction of sequence asymmetric tandem mismatches, the experimental thermodynamic parameters for the 22 previously unmeasured sequence symmetric tandem mismatches are reported. These new data, however, do not improve prediction of the free energy contributions of sequence asymmetric tandem mismatches. Therefore, a new model, independent of sequence symmetric tandem mismatch free energies, is proposed. This model consists of two penalties to account for destabilizing tandem mismatches, two bonuses to account for stabilizing tandem mismatches, and two penalties to account for A-U and G-U adjacent base pairs. This model improves the prediction of asymmetric tandem mismatch free energy contributions and is likely to improve the prediction of RNA secondary structure from sequence.     

Secondary structure model of the Mason-Pfizer monkey virus 5' leader sequence: identification of a structural motif common to a variety of retroviruses.

A stable secondary structure model is presented for the region 3' of the primer-binding site to 130 bases into the gag sequence of the prototype type D retrovirus Mason-Pfizer monkey virus. Using biochemical probing of RNA from this region in association with free energy minimization, we have identified a stem-loop structure in the region, which from other studies has been shown to be important for genomic RNA encapsidation. The structure involves a highly stable stem of five G-C pairs terminating in a heptaloop. Comparison of the Mason-Pfizer monkey virus structure with one predicted for squirrel monkey retrovirus demonstrates an identical stem and a common ACC motif in the loop. Free energy studies of the secondary structure of the 5' regions of eight other retroviruses predict stem loops which have similar GAYC motifs. We believe this may represent a common structural and sequence motif which among other functions may be involved in genomic RNA packaging in these viruses.     

Melting and chemical modification of a cyclized self-splicing group I intron: similarity of structures in 1 M Na+, in 10 mM Mg2+, and in the presence of substrate

C IVS is the cyclized form of the intron from the RNA precursor of the Tetrahymena thermophila large subunit (LSU) ribosomal RNA. C IVS was mapped by chemical modification in 1 M Na+, 0.05 M Na+ and 10 mM Mg2+ (Na+/Mg2+), and Na+/Mg2+ with CUCU substrate. The results suggest the secondary structure is similar for all three conditions. Optical melting curves were also measured for C IVS in 1 M Na+ and Na+/Mg2+ and indicate the secondary structures have similar stabilities under both conditions. Computer predictions of secondary structure and stability are in good agreement with observations. The results suggest that many of the approximations used for computer prediction of secondary structure by free energy minimization are reasonable.     

5S RNA sequence from the Philosamia silkworm: evidence for variable evolutionary rates in insect 5S RNA.

The primary structure of 5S RNA isolated from the posterior silkgland of Philosamia cynthia ricini was determined using three in vitro labelling techniques. The derived sequence consists of 119 nucleotides and can be folded into the secondary structure model proposed for eukaryotic 5S RNAs. This 5S RNA differs from the Bombyx mori molecule in 9 positions and from the Drosophila melanogaster sequence in 14 positions. The comparison of evolutionary rates in insect 5S RNA with inferred rates in other eukaryotic phyla leads to the conclusion that 5S RNA evolution is not constant in different eukaryotic branches, a condition which must be taken into account in phylogenetic tree constructions.     

Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNA^Phe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules.     

Prediction of RNA secondary structure by free energy minimization

RNA secondary structure is often predicted from sequence by free energy minimization. Over the past two years, advances have been made in the estimation of folding free energy change, the mapping of secondary structure and the implementation of computer programs for structure prediction. The trends in computer program development are: efficient use of experimental mapping of structures to constrain structure prediction; use of statistical mechanics to improve the fidelity of structure prediction; inclusion of pseudoknots in secondary structure prediction; and use of two or more homologous sequences to find a common structure.