Statistics of RNA secondary structures

A statistical reference for RNA secondary structures with minimum free energies is computed by folding large ensembles of random RNA sequences. Four nucleotide alphabets are used: two binary alphabets, AU and GC, the biophysical AUGC and the synthetic GCXK alphabet. RNA secondary structures are made of structural elements, such as stacks, loops, joints, and free ends. Statistical properties of these elements are computed for small RNA molecules of chain lengths up to 100. The results of RNA structure statistics depend strongly on the particular alphabet chosen. The statistical reference is compared with the data derived from natural RNA molecules with similar base frequencies. Secondary structures are represented as trees. Tree editing provides a quantitative measure for the distance dt, between two structures. We compute a structure density surface as the conditional probability of two structures having distance t given that their sequences have distance h. This surface indicates that the vast majority of possible minimum free energy secondary structures occur within a fairly small neighborhood of any typical (random) sequence. Correlation lengths for secondary structures in their tree representations are computed from probability densities. They are appropriate measures for the complexity of the sequence-structure relation. The correlation length also provides a quantitative estimate for the mean sensitivity of structures to point mutations.     

Prediction of RNA secondary structure based on helical regions distribution

MOTIVATION: RNAs play an important role in many biological processes and knowing their structure is important in understanding their function. Due to difficulties in the experimental determination of RNA secondary structure, the methods of theoretical prediction for known sequences are often used. Although many different algorithms for such predictions have been developed, this problem has not yet been solved. It is thus necessary to develop new methods for predicting RNA secondary structure. The most-used at present is Zuker's algorithm which can be used to determine the minimum free energy secondary structure. However many RNA secondary structures verified by experiments are not consistent with the minimum free energy secondary structures. In order to solve this problem, a method used to search a group of secondary structures whose free energy is close to the global minimum free energy was developed by Zuker in 1989. When considering a group of secondary structures, if there is no experimental data, we cannot tell which one is better than the others. This case also occurs in combinatorial and heuristic methods. These two kinds of methods have several weaknesses. Here we show how the central limit theorem can be used to solve these problems. RESULTS: An algorithm for predicting RNA secondary structure based on helical regions distribution is presented, which can be used to find the most probable secondary structure for a given RNA sequence. It consists of three steps. First, list all possible helical regions. Second, according to central limit theorem, estimate the occurrence probability of every helical region based on the Monte Carlo simulation. Third, add the helical region with the biggest probability to the current structure and eliminate the helical regions incompatible with the current structure. The above processes can be repeated until no more helical regions can be added. Take the current structure as the final RNA secondary structure. In order to demonstrate the confidence of the program, a test on three RNA sequences: tRNAPhe, Pre-tRNATyr, and Tetrahymena ribosomal RNA intervening sequence, is performed. AVAILABILITY: The program is written in Turbo Pascal 7.0. The source code is available upon request. CONTACT: Wujj@nic.bmi.ac.cn or Liwj@mail.bmi.ac.cn     

Genotyping HIV-1 and HCV strains by a combinatorial DNA melting assay (COMA)

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) strains can be genetically classified into genetic lineages known as genetic types or subtypes according to phylogenetic analyses of complete or partial nucleotide sequences of their genomes. The genetic classification of HIV-1 and HCV strains has important implications for the development of globally effective vaccines and for the management of patients. MATERIALS AND METHODS: A new method, termed combinatorial DNA melting assay (COMA), allows rapid accessing of comparative genetic information between related DNA sequences, making it possible to rapidly and accurately genotype unknown HIV-1 and HCV strains. COMA is mainly based on the differential melting properties of long DNA heteroduplexes. Combinatorial arrays of DNA heteroduplexes are formed when captured PCR-amplified reference DNA with known nucleotide sequences are combined with solution-phase complementary and antigenically labeled DNA with unknown sequences. Genetic divergence between the known and the unknown sequences is inferred as the experimentally derived melting curves of the two strands of the DNA heteroduplexes increasingly diverge. RESULTS: COMA was successfully applied to the genetic classification of HIV-1 and HCV strains into phylogenetic lineages or subtypes. CONCLUSIONS: Use of this assay should accelerate current efforts to understand the global molecular epidemiology of HIV-1 and HCV and may extend to the genetic characterization of other genetically diverse infectious pathogens associated with numerous diseases.     

U1 small nuclear RNA and spliceosomal introns in Euglena gracilis

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote.     

Evolutionary variation in bacterial RNase P RNAs

Sequences encoding RNase P RNAs from representatives of the last remaining classical phyla of Bacteria have been determined, completing a general phylogenetic survey of RNase P RNA sequence and structure. This broad sampling of RNase P RNAs allows some refinement of the secondary structure, and reveals patterns in the evolutionary variation of sequences and secondary structures. Although the sequences range from 100 to <25% identical to one another, and although only 40 of the nucleotides are invariant, there is considerable conservation of the underlying core of the RNA sequence. RNase P RNAs, like group I intron RNAs but unlike ribosomal RNAs, transfer RNAs or other highly conserved RNAs, are quite variable in secondary structure outside of this conserved structural core. Conservative regions of the RNA evolve by substitution of apparently interchangeable alternative structures, rather than the insertion and deletion of helical elements that occurs in the more variable regions of the RNA. In a remarkable case of convergent molecular evolution, most of the unusual structural elements of type B RNase P RNAs of the low G+C Gram-positive Bacteria have evolved independently in Thermomicrobium roseum , a member of the green non-sulfur Bacteria.     

Three-dimensional comparative modeling of RNA

Comparative sequence analysis and ERNA-3D software were used to model the three-dimensional structure of the small domain of signal recognition particle RNA. RNA secondary structures were established by allowing only phylogenetically-supported base pairs. The folding of the RNA molecules was constrained further to include a well-supported pseudoknot. Helical sections were oriented coaxially where a continuous helical stack was formed in the RNA of another species. Finally, RNA helices were placed at distances that preserved the connectivity of the molecule with the smallest number of single-stranded nucleotide residues as identified from the aligned sequences. We show that the comparative three-dimensional structure modeling approach is an extremely powerful tool as it requires only a critical number of carefully aligned sequences.     

Prediction of RNA Secondary Structure Based on Particle Swarm Optimization

A novel method for the prediction of RNA secondary structure was proposed based on the particle swarm optimization(PSO).PSO is known to be effective in solving many different types of optimization problems and known for being able to approximate the global optimal results in the solution space.We designed an efficient objective function according to the minimum free energy,the number of selected stems and the average length of selected stems.We calculated how many legal stems there were in the sequence,and selected some of them to obtain an optimal result using PSO in the right of the objective function.A method based on the improved particle swarm optimization(IPSO)was proposed to predict RNA secondary structure,which consisted of three stages.The first stage was applied to encoding the source sequences,and to exploring all the legal stems.Then,a set of encoded stems were created in order to prepare input data for the second stage.In the second stage,IPSO was responsible for structure selection.At last,the optimal result was obtained from the secondary structures selected via IPSO.Nine sequences from the comparative RNA website were selected for the evaluation of the proposed method.Compared with other six methods,the proposed method decreased the complexity and enhanced the sensitivity and specificity on the basis of the experiment results.     

Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA

Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.     

RNA aptamers to the peptidyl transferase inhibitor chloramphenicol

BACKGROUND: The problem of how macromolecules adopt specific shapes to recognize small molecules in their environment is readily addressed through in vitro selections (the SELEX protocol). RNA-antibiotic interactions are particularly attractive systems for study because they provide an opportunity to expand our understanding of molecular recognition by RNA and to facilitate ribosomal modeling. Specifically, the antibiotic chloramphenicol (Cam) naturally binds bacterial ribosomes in the 'peptidyl transferase loop' of 23S ribosomal RNA to inhibit peptide bond formation. RESULTS: We identified Cam-binding RNA molecules ('aptamers') from two independent initial random RNA populations. Boundary determinations, ribonuclease S1 sensitivity analyses and the activity of truncated minimal RNAs identified a structural motif that is shared by sequences from both selections. The pseudosymmetric motif consists of a highly conserved central helix of five to six base pairs flanked by A-rich bulges and additional helices. Addition of Cam prior to ribonuclease S1 protected nucleotides in the conserved cores from cleavage. Reselection from a pool of mutated variants of the minimal aptamer further refined the sequence requirements for binding. Finally, we used proton nuclear magnetic resonance (NMR) to establish a 1:1 RNA: Cam stoichiometry of the complex. Both the protection and NMR data both show that Cam stabilizes the active fold of this aptamer. CONCLUSIONS: There are many different RNA sequences that can bind Cam. The Cam aptamers that we examined have a well-defined secondary structure with a binding pocket that appears to be stabilized by Cam. This RNA motif superficially resembles the Cam-binding site in 23S rRNA, although further work is needed to establish the significance of these similarities.     

Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5' and 3' of the catalytic site

In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with polyadenosine sequences and to a lesser degree diadenosines and monoadenosine residues. Pausing was also inversely proportional to the potential bond strength between the nascent strand and the template at the point of termination, for both mono and dinucleotides. A run of five adenosine and four uridine residues caused most pausing on the HIV-1 template, a region which is the site of much sequence heterogeneity in HIV-1. We propose that homopolyadenosine tracts can act as termination signals for RT in the context of inverted repeats as they do for certain RNA polymerases.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards

The widespread use of sensitive assays for the detection of viral and cellular RNA sequences has created a need for stable, well-characterized controls and standards. We describe the development of a versatile, novel system for creating RNase-resistant RNA. "Armored RNA" is a complex of MS2 bacteriophage coat protein and RNA produced in Escherichia coli by the induction of an expression plasmid that encodes the coat protein and an RNA standard sequence. The RNA sequences are completely protected from RNase digestion within the bacteriophage-like complexes. As a prototype, a 172-base consensus sequence from a portion of the human immunodeficiency virus type 1 (HIV-1) gag gene was synthesized and cloned into the packaging vector used to produce the bacteriophage-like particles. After production and purification, the resulting HIV-1 Armored RNA particles were shown to be resistant to degradation in human plasma and produced reproducible results in the Amplicor HIV-1 Monitor assay for 180 days when stored at -20 degreesC or for 60 days at 4 degreesC. Additionally, Armored RNA preparations are homogeneous and noninfectious.