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Targeting G- quadruplex DNA ligand screening methods

Author: QiaoYunXia
Tutor: JinYan
School: Shaanxi Normal University
Course: Analytical Chemistry
Keywords: telomere DNA G-quadruplex-binding ligands electrochemical fluorescence daunorubicin gold nanoparticles
CLC: R96
Type: Master's thesis
Year: 2011
Downloads: 129
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Abstract

G-quadruplex formation can effectively inhibit telomere maintainance. Therefore, ligands of G-quadruplex have received great attention. The study of interaction mechanism between antitumor drug and different DNAs is very interesting and significant in the development of a new antitumor drug. In this thesis, electrochemical and fluorescence methods have been developed for selecting quadruplex-binding ligands based on structure switching of human telomere caused by the specific binding between human telomere and anti-cancer drugs using anthracycline antibiotic and alkaloid with antiblastic activity as model ligands. In addition, the interaction mode and affinity of daunorubicin (DNR) with ssDNA, dsDNA and quadruplex DNA have been systematically studied by electrochemical and fluorescence measurements. The contents and results of research in this thesis contain the following three parts:1. A label-free method for identifying electroactive G-quadruplex-binding ligandA label-free electrochemical method has been developed to identify electrochemically active molecules that stabilize G-quadruplexes in homogeneous medium. Daunorubicin (DNR) was used as model drug. In the presence of GDNA, the voltammetry of DNR changed. The binding constant (P) and binding-site number (m) of GDNA-mDNR are simultaneously obtained from dependence of the current on the amount of added GDNA in voltammetry, concluding thatβ=(2.37±2.25)×107 L/mol and m was near I, respectively. Control experiment was performed, indicating that this method can specifically identify G-quadruplex-binding ligand. The interaction mechanism of DNR with GDNA was also investigated by circular dichroizm. The combined data from them demonstrated that the electrochemical approach offers a simple and effective method to identify electroactive ligand with potential anticancer activity.2. The studying of binding mechanism and affinity between daunorubicin and different DNAsHere, we make a systematic study of the interaction mode and affinity of daunorubicin (DNR) with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and quadruplex DNA. Cyclic voltammograms of DNR at the bare glassy carbon electrode (GCE) and at the DNA modified GCE were compared to study the binding mode of DNR with different DNAs. UV-Vis absorption spectral and fluorescence polarization studies were also used to investigate the interaction mechanism of daunorubicin and human telomere DNA (GDNA). The binding affinity of DNR with different DNAs was studied by cyclic voltammetry assay and fluorescence titration in homogeneous solution. Combined data indicated that DNR has higher affinity and specificity for quadruplex DNA and dsDNA than ssDNA.3. Fluorescence resonance energy transfer assay for identifying G-Quadruplex-binding ligands using DNA functionalized gold nanoparticles as fluorescence quencherA fluorescence resonance energy transfer (FRET) system has been developed for identifying molecules that stabilize G-quadruplexes in homogeneous medium using DNA functionalized gold nanoparticles (GNPs) as fluorescence quencher and FAM-tagged human telomeric sequence (F-GDNA) as recognition probe. Thiolated complementary strand of human telomere DNA (cDNA) functionalized the GNPs surface, and then hybridized with F-GDNA, resulting in the fluorescence quenching of F-GDNA (or FAM) by GNPs. However, the fluorescence restored when single-stranded F-GDNA folded into a G-quadruplex structure upon quadruplex ligands binding, leading to release F-GDNAs from the surface of GNPs. G-quadruplex formation induced by specific binding of GDNA ligands was investigated by CD measurements. The combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to fold single-stranded F-GDNA into G-quadruplex DNA. It is a simple and effective approach to identify G-quadruplex-binding ligands.

Targeting G- quadruplex DNA ligand screening methods

Abstract

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