Frequency of the occurrence of the hydrogen bonds.

<p>Frequency of the occurrence of the hydrogen bonds (HBs), monitored over 2 ns, every 10 ps, in the guanine core in the presence of C3 and A1T3 mutated G-quadruplex sequences. </p

RMSd conformation of C3 and A1T3 mutated G-quadruplex sequences.

<p>Lowest (A) and highest (B) RMSd conformation of C3 and A1T3 mutated G-quadruplex sequences during 2 ns MD simulations with respect to the starting structure (2HY9). The DNA is shown in purple (C3) and aquamarine (A1T3) wireframe rendering and its strand as orange cartoon. The K⁺ coordinating ions are represented as blue spheres. The RMSd value of each conformation is reported and expressed in Å. </p

CD spectra of telomeric oligonucleotides mutated in the loop.

<p>In each oligonucleotide one single base was mutated. </p

CD spectra of telomeric oligonucleotides mutated in the loop.

<p>A) Each oligonucleotide contained one single base mutated to C in each loop; B) each oligonucleotide contained the three nucleotides mutated to C in one loop; C) A bases in the loops were moved from the third to the second and first positions or mutated to T.</p

Thermal difference spectra (TDS) and TDS factor plots of three representative oligonucleotides.

<p>A) TDS of C1, C2 and C3 sequences; B) TDS factors of C1, C2 and C3 sequences. TDS and TDS factors of all oligonucleotides are available in Figure S8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084113#pone.0084113.s004" target="_blank">File S4</a>.</p

Model structures of conformations reported for the human telomeric sequence.

<p>Guanosines in dark grey are <i>syn</i>, and those in light gray are <i>anti</i>.</p

Clerocidin protection assay of the wt, C1, C2 and C3 telomeric oligonucleotides.

<p>Sequences were heat denatured, folded in the presence or absence of K⁺ and treated with CL followed by hot piperidine (CL lanes) or just treated with piperidine (C lanes). M indicates the marker lane obtained with the Maxam and Gilbert protocol. Base sequences are shown aside each gel image. Symbols * and ~ indicate protected G and C bases, respectively.</p

CD-monitored thermal denaturing assays of wt, C1, C2 and C3 telomeric oligonucleotides.

<p>A-D) CD spectra of each indicated oligonucleotide measured at increasing temperatures (20°C-95°). Arrows indicate curve trend at increasing temperatures. E) Molar ellipticities, measured at wavelength of maximum intensity, were plotted against temperature.</p

Identification and Characterization of New DNA G‑Quadruplex Binders Selected by a Combination of Ligand and Structure-Based Virtual Screening Approaches

Nowadays, it has been demonstrated that DNA G-quadruplex arrangements are involved in cellular aging and cancer, thus boosting the discovery of selective binders for these DNA secondary structures. By taking advantage of available structural and biological information on these structures, we performed a high throughput in silico screening of commercially available molecules databases by merging ligand- and structure-based approaches by means of docking experiments. Compounds selected by the virtual screening procedure were then tested for their ability to interact with the human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and photodynamic techniques. Interestingly, our screening succeeded in retrieving a new promising scaffold for G-quadruplex binders characterized by a psoralen moiety

HCV Genotypes Are Differently Prone to the Development of Resistance to Linear and Macrocyclic Protease Inhibitors

<div><h3>Background</h3><p>Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs).</p> <h3>Methods</h3><p>The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed.</p> <h3>Results</h3><p>Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <u><</u>1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T).</p> <h3>Conclusions</h3><p>The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.</p> </div