13 research outputs found
IC<sub>50</sub> values against Telomerase found for the different lgands by TTAP-LIG assay<sup>a</sup>.
a<p>The results are average of three experiments and are within ±1% of each other.</p
Melting temperatures<sup>a</sup> of Hum<sub>24</sub> and Hum<sub>48</sub> G4DNAs formed in (Na + LiCl)<sup>b</sup> and NaCl<sup>c</sup> solutions and G4DNA-ligand complexes ([ligand]:[DNA] ratio ‘r’ = 20 for M and 10 for the gemini ligands respectively).
a<p>Changes in the circular dichroism spectral peak at 295 nm monitored as a function of temperature using 2 µM strand ODN concentration. <b><sup>b</sup></b>(Na <b>+</b> LiCl solution)  =  (10 mM sodium cacodylate pH 7.4 having 100 mM LiCl and 0.1 mM EDTA). <b><sup>c</sup></b>NaCl solution  =  (10 mM Tris-HCl, pH 7.4 having 100 mM NaCl and 0.1 mM EDTA). <b><sup>d</sup></b>Δ<i>T</i><sub>m</sub> values were obtained from the difference in the melting temperatures of the ligand bound and uncomplexed G4DNA. <b><sup>e</sup></b>No significant increment in <i>T</i><sub>m</sub>. <b><sup>f</sup></b>For entry 11, the G4DNA was formed in LiCl buffer (first heated to 95°C for 5 min and then cooled slowly). The results are average of two experiments and are within ±0.5°C of each other.</p
Stabilization and Structural Alteration of the G‑Quadruplex DNA Made from the Human Telomeric Repeat Mediated by Tröger’s Base Based Novel Benzimidazole Derivatives
Ligand-induced stabilization of the G-quadruplex DNA
structure
derived from the single-stranded 3′-overhang of the telomeric
DNA is an attractive strategy for the inhibition of the telomerase
activity. The agents that can induce/stabilize a DNA sequence into
a G-quadruplex structure are therefore potential anticancer drugs.
Herein we present the first report of the interactions of two novel
bisbenzimidazoles (<b>TBBz1</b> and <b>TBBz2</b>) based
on Tröger’s base skeleton with the G-quadruplex DNA
(G4DNA). These Tröger’s base molecules stabilize the
G4DNA derived from a human telomeric sequence. Evidence of their strong
interaction with the G4DNA has been obtained from CD spectroscopy,
thermal denaturation, and UV–vis titration studies. These ligands
also possess significantly higher affinity toward the G4DNA over the
duplex DNA. The above results obtained are in excellent agreement
with the biological activity, measured in vitro using a modified TRAP
assay. Furthermore, the ligands are selectively more cytotoxic toward
the cancerous cells than the corresponding noncancerous cells. Computational
studies suggested that the adaptive scaffold might allow these ligands
to occupy not only the G-quartet planes but also the grooves of the
G4DNA
Simulated structures of the dimeric G4DNA-gemini ligand complexes and topology change of the G4DNA.
<p>Simulated structures of (A) <b>D2</b>-dimeric G4DNA complex and (B) <b>D3</b>-dimeric G4DNA complex are shown as a ‘stick’ model. In the case of <b>D2</b>, both bisbenzimidazole units bind with the grooves of different G4 DNA units. In the case of <b>D3</b>, one bisbenzimidazole stacks on the 5′-surface of the G-quartet plane while the other bisbenzimidazole binds to the groove of the G-quadruplex. K<sup>+</sup> ions present in the central cavity are shown in cyan colour. Ligand is shown in sticks model colored by atom type (magenta-blue). Hybrid G4DNA is represented as a cartoon form (green-orange). (C and D) Proposed structures of the dimeric G4DNA formed from longer human telomeric repeats: C) hybrid dimeric G4DNA (dhyq) formed in K<sup>+</sup> solution; D) parallel dimeric G-quadruplex DNA (dpq) formed in K<sup>+</sup> solution in presence of various ligands (<b>M</b> or <b>D</b>s).</p
Melting temperatures (<i>T</i><sub>m</sub>)<sup>a</sup> of Hum<sub>24</sub> and Hum<sub>48</sub> G4DNA, ligand bound to pre-formed G4DNA and complexes produced when G4DNA was formed in presence of the indicated ligand at [ligand]:[DNA] ratio ‘r’ = 20 for the monomeric and ‘r’ = 10 for the gemini ligands in 100 mM KCl solution.
a<p>Changes in the circular dichroism spectral peak (at 295 nm for the G4DNA and the pre-formed G4DNA-ligand complex; at 266 nm for the G4DNA-ligand complex formed in presence of ligand) monitored as a function of temperature using 2 µM strand ODN concentration. <b><sup>b</sup></b>Δ<i>T</i><sub>m</sub> values were obtained from the difference in the melting temperatures of the ligand bound and uncomplexed G4DNA. For the G4DNA-ligand complexes when G4DNA was formed in presence of the ligand, the solution was first heated to 95°C for 5 min and then cooled slowly. The results are average of two experiments and are within ±0.5°C of each other.</p
Selective toxicity of ligands toward cancer cells.
<p>Effect of the ligands on the cell viability after 48 h exposure of HEK293 (normal) and HEK293T (cancerous) cells with each ligand at different concentrations as measured by MTT (methyl thiazolyl tetrazolium) assay. Each experiment was repeated six times.</p
CD spectra of the G4DNA without ligands and CD titrations in KCl solution.
<p>Circular dichroic (CD) spectral profiles of G4DNA formed using the Hum<sub>24</sub> (Panel A) and the Hum<sub>48</sub> (Panel B), 4 µM DNA in each case, in presence of the indicated monovalent cations (100 mM). (Panels C and D) CD spectral titrations of the pre-formed G4DNA (4 µM strand concn.) formed using the Hum<sub>48</sub> in KCl buffer (10 mM Tris, pH 7.4 having 100 mM KCl) in presence of <b>M</b> (Panel C) and <b>D3</b> (Panel D) at ligand: DNA ratio (r) = 10, 20, 30 (for <b>M</b>) and r = 5, 10, 15, 20 (for <b>D3</b>). Arrows indicate the direction of increments observed in the CD intensity.</p
Topology of the G4DNA and the ligands used in the study.
<p>(A-C) Folding topology of the monomeric intramolecular G4DNAs as elucidated from NMR or X-ray crystallography: A) basket-type intramolecular G4DNA in Na<sup>+</sup> solution (NMR) B) hybrid-1 intramolecular G4DNA in K<sup>+</sup> solution (NMR) and C) propeller-type parallel-stranded intramolecular G4DNA in presence of K<sup>+</sup> in the crystalline state (X-ray crystal structure). (D) Chemical structures of the ligands used in the present study.</p
Telomerase inhibitory properties of ligands by TRAP-LIG assay.
<p>TRAP-LIG assay was performed using indicated concentrations over each lane with <b>M</b> and <b>D1</b> (A); <b>D2</b> and <b>D3</b> (B). A positive control was run with telomerase, but with no ligand. A negative control was run without either telomerase or ligand. Positive and negative control lanes are indicated by + and – labels, respectively. Other lanes contain TRAP reaction mixtures mixed with the indicated concentrations (µM) of each ligand.</p
N‑Terminal Disordered Domain of <i>Saccharomyces cerevisiae</i> Hop1 Protein Is Dispensable for DNA Binding, Bridging, and Synapsis of Double-Stranded DNA Molecules But Is Necessary for Spore Formation
The
cytological architecture of the synaptonemal complex (SC),
a meiosis-specific proteinaceous structure, is evolutionarily conserved
among eukaryotes. However, little is known about the biochemical properties
of SC components or the mechanisms underlying their roles in meiotic
chromosome synapsis and recombination. Functional analysis of <i>Saccharomyces cerevisiae</i> Hop1, a key structural component
of SC, has begun to reveal important insights into its function in
interhomolog recombination. Previously, we showed that Hop1 is a structure-specific
DNA-binding protein, exhibits higher binding affinity for the Holliday
junction, and induces structural distortion at the core of the junction.
Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular
synapsis between duplex DNA molecules. Here, we show that Hop1 possesses
a modular domain organization, consisting of an intrinsically disordered
N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD).
Furthermore, we found that Hop1CTD exhibits strong homotypic as well
as heterotypic protein–protein interactions, and its biochemical
activities were similar to those of the full-length Hop1 protein.
However, Hop1CTD failed to complement the meiotic recombination defects
of the <i>Δhop1</i> strain, indicating that both N-
and C-terminal domains of Hop1 are essential for meiosis and spore
formation. Altogether, our findings reveal novel insights into the
structure–function relationships of Hop1 and help to further
our understanding of its role in meiotic chromosome synapsis and recombination