RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF), and one set describes large conformational changes between ligand-free and ligand-bound states. The Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal a notable need for an algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations.

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson-Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.

RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

Complete genome sequencing and genetic characterization of Alkhumra hemorrhagic fever virus isolated from Najran, Saudi Arabia.

BACKGROUND: Alkhumra hemorrhagic fever virus (AHFV) is a newly described flavivirus first isolated in 1994-1995 from the Alkhumra district south of Jeddah, Saudi Arabia. Subsequently, the virus was also isolated from Makkah (2001-2003) and Najran (2008-2009), Saudi Arabia. METHODS: The full-length genome of an AHFV strain isolated from patients in Najran (referred to as AHFV/997/NJ/09/SA) was PCR amplified and sequenced, and compared with the sequences of 18 other AHFV strains previously isolated from Jeddah and Makkah, dengue virus (DENV), Kyasanur forest disease virus (KFDV), Langat virus, Omsk hemorrhagic fever virus (OHFV), and tick-borne encephalitis virus (TBEV). RESULTS: The RNA of the AHFV/997/NJ/09/SA strain was found to have 10,546 nucleotides encoding for a single 3,416-amino acid polyprotein, whereas the previously reported AHFV strains were composed of 10,685-10,749 nucleotides. The AHFV/997/NJ/09/SA strain showed about 99% homology with the previously reported AHFV strains. The KFDV, Langat virus, TBEV, and OHFV isolates formed a separate cluster with a variable homology. The most important variations were observed in the core protein and NS4a gene sequences of two AHFV isolates. CONCLUSION: The variation in the number of nucleotides and phylogenetic analysis with the other AHFV isolates could have resulted from recombination of circulating virus strains.

Solution structure of RNase P RNA.

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes.

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°(37), ΔH° and melting temperature (T(m)) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5' of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3' of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.

Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

Measurement of Klebsiella Intestinal Colonization Density To Assess Infection Risk.

Klebsiella pneumoniae and the closely related species K. variicola and K. quasipneumoniae are common causes of health care-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed. A multiplex quantitative PCR (qPCR) assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and a preceding or coincident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. qPCR from rectal swab samples was used to measure the association between the relative abundance of Klebsiella and subsequent infections. The Klebsiella relative abundance by qPCR was highly correlated with 16S sequencing (ρ = 0.79; P < 0.001). The median Klebsiella relative abundance was higher in cases (15.7% [interquartile range {IQR}, 0.93 to 52.6%]) (n = 83) than in controls (1.01% [IQR, 0.02 to 12.8%]) (n = 155) (P < 0.0001). Adjusting for multiple clinical covariates using inverse probability of treatment weighting, a Klebsiella relative abundance of >22% was associated with infection overall (odds ratio [OR], 2.87 [95% confidence interval {CI}, 1.64 to 5.03]) (P = 0.0003) and with bacteremia in a secondary analysis (OR, 4.137 [95% CI, 1.448 to 11.818]) (P = 0.0084). Measurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection. IMPORTANCE Colonization by bacterial pathogens often precedes infection and offers a window of opportunity to prevent these infections in the first place. Klebsiella colonization is significantly and reproducibly associated with subsequent infection; however, factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, a high Klebsiella density, defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available PCR technology, this type of assay could be deployed in clinical laboratories to identify patients at an increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention intervention.

Hachimoji DNA and RNA: A genetic system with eight building blocks.

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Pseudouridines in rRNA helix 69 play a role in loop stacking interactions.

The (1)H NMR spectra of RNAs representing E. coli 23S rRNA helix 69 with [1,3-(15)N]pseudouridine modification at specific sites reveal unique roles for pseudouridine in stabilizing base-stacking interactions in the hairpin loop region.

Physical principles and visual-OMP software for optimal PCR design.

The physical principles of DNA hybridization and folding are described within the context of how they are important for designing optimal PCRs. The multi-state equilibrium model for computing the concentrations of competing unimolecular and bimolecular species is described. Seven PCR design "myths" are stated explicitly, and alternative proper physical models for PCR design are described. This chapter provides both a theoretical framework for understanding PCR design and practical guidelines for users. The Visual-OMP (oligonucleotide modeling platform) package from DNA Software, Inc. is also described.

Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes.

Nearest-neighbor thermodynamic parameters of the 'universal pairing base' deoxyinosine were determined for the pairs I.C, I.A, I.T, I.G and I.I adjacent to G.C and A.T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the T(m) for all sequences within 1.2 degrees C on average. The general trend in decreasing stability is I.C > I.A > I.T approximately I. G > I.I. The stability trend for the base pair 5' of the I.X pair is G.C > C.G > A.T > T.A. The stability trend for the base pair 3' of I.X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes.

Investigation of the Yamaguchi esterification mechanism. Synthesis of a lux-s enzyme inhibitor using an improved esterification method.

[reaction: see text] A one-pot procedure for the regioselective synthesis of aliphatic esters is described. This was a result of a study on mixed aliphatic-aromatic anhydrides. The data suggest that during the Yamaguchi esterification reaction, a symmetric aliphatic anhydride is produced in situ, which upon reaction with an alcohol yields the ester. We confirmed that benzoyl chloride could be used instead of the sterically hindered Yamaguchi acid chloride. This method was successfully applied in the synthesis of Lux-S aspartic acid inhibitor.

Binding of specific DNA base-pair mismatches by N-methylpurine-DNA glycosylase and its implication in initial damage recognition.

Most DNA glycosylases including N-methylpurine-DNA glycosylase (MPG), which initiate DNA base excision repair, have a wide substrate range of damaged or altered bases in duplex DNA. In contrast, uracil-DNA glycosylase (UDG) is specific for uracil and excises it from both single-stranded and duplex DNAs. Here we show by DNA footprinting analysis that MPG, but not UDG, bound to base-pair mismatches especially to less stable pyrimidine-pyrimidine pairs, without catalyzing detectable base cleavage. Thermal denaturation studies of these normal and damaged (e.g. 1,N(6)-ethenoadenine, varepsilonA) base mispairs indicate that duplex instability rather than exact fit of the flipped out damaged base in the catalytic pocket is a major determinant in the initial recognition of damage by MPG. Finally, based on our determination of binding affinity and catalytic efficiency we conclude that the initial recognition of substrate base lesions by MPG is dependent on the ease of flipping of the base from unstable pairs to a flexible catalytic pocket.

Photoinduced cleavage by a rhodium complex at G.U mismatches and exposed guanines in large and small RNAs.

Photoinduced cleavage reactions by the rhodium complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)(3)(3+)] with three RNA hairpins, r(GGGGU UCGCUC CACCA) (16 nucleotide, tetraloop(Ala2)), r(GGGGCUAUAGCUCUAGCUC CACCA) (24 nucleotide, microhelix(Ala)), and r(GGCGGUUAGAUAUCGCC) (17 nucleotide, 790 loop), and full-length (1542 nucleotide) 16S rRNA from Escherichia coli were investigated. The cleavage reactions were monitored by gel electrophoresis and the sites of cleavage by Rh(DIP)(3)(3+) were determined by comparisons with chemical or enzymatic sequencing reactions. In general, RNA backbone scission by the metal complex was induced at G.U mismatches and at exposed G residues. The cleavage activity was observed on the three small RNA hairpins as well as on the isolated 1542-nucleotide ribosomal RNA.

The synthesis of anti-fixed 3-methyl-3-deaza-2'-deoxyadenosine and other 3H-imidazo[4,5-c]pyridine analogs.

Rotation of a heterocyclic base around a glycosidic bond allows the formation of syn and anti conformations in nucleosides. The syn conformation has been observed primarily in purine-purine mismatches in DNA duplexes. Such mismatches give rise to false positive oligonucleotide hybridization in DNA-based diagnostics. Here we describe the synthesis of an analog of 2'-deoxyadenosine that retains its Watson-Crick functional groups, but cannot form the syn conformation. In this analog, the N3 atom of 2'-deoxyadenosine is replaced by a C-CH3 group to give 7-methyl-1-beta-D-deoxyribofuranosyl-1H-imidazo[4,5-c]pyridin-4-ylamine or 3-methyl-3-deaza-2'-deoxyadenosine (3mddA). This modification sterically prevents the syn conformation and 3mddA becomes an anti-fixed nucleoside analog of 2'-deoxyadenosine. The synthesis and conformational analysis of 3mddA and several analogs with an 3H-imidazo[4,5-c]pyridine skeleton are described, as well as their potential applications.

Comparison of solution conformations and stabilities of modified helix 69 rRNA analogs from bacteria and human.

The helix 69 (H69) region of the large subunit (28S) ribosomal RNA (rRNA) of Homo sapiens contains five pseudouridine (Ψ) residues out of 19 total nucleotides, three of which are highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine in double-stranded (stem) regions. These results were compared with previous hairpin (stem plus single-stranded loop) studies to understand the contributions of the loop sequences to H69 structure and stability. The role of a loop nucleotide substitution from an A in bacteria (position 1918 in Escherichia coli 23S rRNA) to a G in eukaryotes (position 3734 in H. sapiens 28S rRNA) was examined. Thermodynamic parameters for the duplex RNAs were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by circular dichroism spectroscopy. The overall folded structure of human H69 appears to be similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved; however, our results reveal subtle differences in structure and stability between the bacterial and human H69 RNAs in both the stem and loop regions. These findings may be significant with respect to H69 as a potential drug target site.

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA.

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m(2)G966 and m(5)C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m(2)G966 or m(5)C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.