The Transition from Unfolded to Folded G-Quadruplex DNA Analyzed and Interpreted by Two-Dimensional Infrared Spectroscopy

A class of DNA folds/structures known collectively as G-quadruplexes (G4) commonly forms in guanine-rich areas of genomes. G4-DNA is thought to have a functional role in the regulation of gene transcription and telomerase-mediated telomere maintenance and, therefore, is a target for drugs. The details of the molecular interactions that cause stacking of the guanine-tetrads are not well-understood, which limits a rational approach to the drugability of G4 sequences. To explore these interactions, we employed electron-vibration-vibration two-dimensional infrared (EVV 2DIR) spectroscopy to measure extended vibrational coupling spectra for a parallel-stranded G4-DNA formed by the Myc2345 nucleotide sequence. We also tracked the structural changes associated with G4-folding as a function of K+-ion concentration. To classify the structural elements that the folding process generates in terms of vibrational coupling characteristics, we used quantum-chemical calculations utilizing density functional theory to predict the coupling spectra associated with given structures, which are compared against the experimental data. Overall, 102 coupling peaks are experimentally identified and followed during the folding process. Several phenomena are noted and associated with formation of the folded form. This includes frequency shifting, changes in cross-peak intensity, and the appearance of new coupling peaks. We used these observations to propose a folding sequence for this particular type of G4 under our experimental conditions. Overall, the combination of experimental 2DIR data and DFT calculations suggests that guanine-quartets may already be present before the addition of K+-ions, but that these quartets are unstacked until K+-ions are added, at which point the full G4 structure is formed

Identification of a novel enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases.

In this study, a virus strain designated as HY12 was isolated from cattle with a disease of high morbidity and mortality in Jilin province. Biological and physiochemical properties showed that HY12 isolates is cytopathic with an extremely high infectivity. HY12 is resistant to treatment of organic solvent and acid, and unstable at 60°C for 1 h. Electron microscopy observation revealed the virus is an approximately 22-28 nm in diameter. The complete genome sequence of HY12 consists of 7416 nucleotides, with a typical picornavirus genome organization including a 5'-untranslated region (UTR), a large single ORF encoding a polyprotein of 2176 amino acids, and a 3'-UTR. Phylogenetic analysis clustered HY12 isolates to a new serotype/genotype within the clade of enterovirus E (formerly BEV-A). Alignment analysis revealed a unique insertion of 2 amino acid residues (NF) at the C-terminal of VP1 protein between aa 825 and 826, and several rare mutations in VP1 and VP4 of HY12 isolates in relation to known bovine enterovirus (BEV) strains. This is the first report of an enterovirus E in China, which is potentially associated with an outbreak in cattle with severe respiratory and enteric diseases

The Transition from Unfolded to Folded G-Quadruplex DNA Analysed and Interpreted by Two-Dimensional Infrared Spectroscopy

A class of DNA folds/structures known collectively as G-quadruplexes (G4) commonly form in guanine-rich areas of genomes. G4 DNA is thought to have a functional role in the regulation of gene transcription and telomerase-mediated telomere maintenance and is therefore a target for drugs. The details of the molecular interactions that cause stacking of the guanine tetrads are not well-understood, which limits a rational approach to the drugability of G4 sequences. To explore these interactions further, we employed Electron-Vibration-Vibration two-dimensional Infra-red (EVV 2DIR) spectroscopy to measure extended vibrational coupling spectra for a parallel-stranded G-quadruplex DNA formed by Myc2345 nucleotide sequence. We also tracked the structural changes associated with G4 folding as a function of K+ ion concentration to generate further insight. In order to classify the structural elements that the folding process generates in terms of vibrational coupling characteristics, we used quantum-chemical calculations utilising density functional theory. This resulted in predictions of the coupling spectra associated with a given structure, which are compared against the experimental coupling data obtained from the EVV 2DIR spectroscopy. Overall, 102 coupling peaks are experimentally identified and followed during the folding process. A number of phenomena are noted and associated with the formation of the folded form. This includes frequency shifting, changes in cross-peak intensity and the appearance of new coupling peaks. The new peaks can be assigned to the coupling between specific chemical groups within the complex, and we use the 2DIR data to propose a folding sequence for this particular type of G4 under our experimental conditions. Overall, the combination of experimental 2DIR data and DFT calculations suggests that guanine quartets may already be present in the initial DNA before the addition of potassium ions, but that these quartets are unstacked until potassium ions are added, at which point the full G4 structure is formed

Partial physiochemical properties for HY12 strain.

<p>Partial physiochemical properties for HY12 strain.</p

Primers used for amplifying the complete genome sequence for HY12.

<p>S stands for sense; AS refers to antisense.</p

Primers used for differentiation of the potential agents.

<p>BPV: bovine parvovirus; FMDV: foot and mouth disease virus; BEV: bovine enterovirus; S: sense; AS: antisense.</p

Recombination revealed in the HY12 strain.

<p>Neighbor-joining trees of the structural proteins VP1-VP4, and the non-structural protein 3D of 15 enteroviruses were compared. When amino acid sequences for VP1, VP3, and VP4 were used to generate phylogenetic tree, similar patterns were observed as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097730#pone-0097730-g003" target="_blank">Fig 3B, 3D, and 3E</a>, indicating a interserotype recombination for the HY12 strain. Like the observation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097730#pone-0097730-g003" target="_blank">Fig 3A, F and G</a>, the HY12 was clustered closely to K2577, SL305, PS 42 and PS 83 strains, an indication of complex interserotypic and intraserotypic recombination in the evolution for HY12. The positions of HY12 were highlighted with a triangle.</p

Unique amino acid mutations in the capsid proteins encoded by HY12 isolates.

<p>The amino acid sequence of HY12 isolates were deduced from the nucleotide sequence, and was aligned with 12 known BEV strains in the GenBank. Alignment analysis was performed using each HY12-encoded structural protein as a template. Results were shown respectively for VP1 (A), VP2 (B), VP3 (C), and VP4 (D). The identical amino acids were marked with symbol “·”, and different amino acids to HY12 were presented as individual amino acid symbol. The unique mutation for HY12 was highlighted with red color. Deletion of amino acids were marked as “-”.</p

Phylogenetic analysis clustered HY12 strain to a new serotype/genotype within enterovirus E.

<p>Phylogenetic tree were generated by neighbor-joining methods by comparing the sequence regions of 5′-UTR, VP1, VP2, VP3, VP4, 3D, and the complete genome sequences for 15 enteroviruses. HY12 strain was placed to the cluster of enteroviruses E after phylogenetic analysis with the all nucleotide sequence regions except 5′-UTR (B–F). The HY12 strain was revealed as a new serotype/genotype (serotype/genotype 3) that only consists of D14/3/96 and HY12 strains in relation to serotype 1 (LC-R4, VG5-27,Vir 404/03) and serotype 2 (SL305, K2577,PS 42, PS 83) enterovirus strains (B–E). When nucleotide sequences for the non-structural proteins 3D and the complete genome sequence were employed, the HY12 were clustered to the same clade most close to SL305 and K2577 within enteroviruses E (F). However, HY12 strain was clustered to neither clade in enteroviruses E nor enterovirus F using the 5′-UTR sequence (A), suggesting an intraserotypic recombination during HY12 evolution. The position of HY12 was highlighted with a triangle.</p

Cytopathic effect of HY12 in cell culture and EM observation.

<p>HY12 caused a typical cytopathic effect in MDBK cells after 6–8 h inoculation. Cells became rounded with an increased refraction (B). 24∼48 h post infection, majority of the infected cells detached off the flask (C). The normal MDBK cells were used as negative controls (A). HY12 virus particles observed by electron microscopy to be about 22∼28 nm in diameter as indicated by arrow (D), the scale bar is 100 nm.</p