||Binding Analysis of Novel Cyclic Naphthalene Diimide with DNA
Islam, Md. Monirul
In recent times, DNA structures are one of the attractive research targets becauseof their vital biological roles and remarkable therapeutic applications. Specially, GquadruplexDNA structures are a very interesting target because of promoter regions,human telomeres and aptemers are closely associated with the genetic integrity, cellproliferation, aging and cancer. Fundamental understanding of the interactions of smallmoleculeligands or proteins with DNA sequences and their structural effect, bindingaffinities, thermodynamic, kinetic and thermal stability are very crucial. This doctoralthesis has uncovered the interaction studies of novel cyclic naphthalene diimidederivatives (cNDIs, 1,2) & non-cyclic naphthalene diimide (NDI 3) with double strandedDNA (dsDNA) such as calf thymus DNA (CT-DNA), poly[d(A-T)]2, or poly[d(G-C)]2and G-quadruplexes DNA such as human telomere DNA (a-core & a-coreTT), promoterregion’s DNA (c-kit & c-myc) and thrombin-binding aptamer (TBA).Firstly, I have studied the interaction of newly synthesized cNDIs 1,2 with varioustypes of dsDNA to observe the cyclic linker chain effect binding to dsDNA. Secondly, Ihave studied the interaction of 2 with various types of G-quadruplexes DNA to observethe specific binding to G-quadruplexes DNA structure. I have studied the interaction ofNDI 3 with dsDNA and G-quadruplexes DNA as a control.The interaction of three different types of dsDNA and seven different types G-richoligonucleotides studied with newly synthesize 1,2 and NDI 3 by the physicochemicaland biochemical method such as UV-Vis spectroscopy, Circular dichroism (CD)spectroscopy, Topoisomerase I assay, Stopped-flow kinetics, Thermal melting studies,TRAP assay and FRET-melting assay experiments.The binding studies between cNDIs 1,2 and DNA duplexes showed affinities inthe order of 105?106 M-1 using UV-Vis spectroscopic titration with a stoichiometry of onecNDI molecule covered four DNA base pairs as a bis-threading intercalation mode ofbinding. The induced CD signal was observed on cNDIs chromophore upon the additionof CT-DNA. Topo I isomerase assay showed that 2 can unwind circular dsDNA, it’s alsoindicated the bis-intercalation binding of cNDIs with dsDNA. According to the van’t Hoffand Gibbs free energy equation, thermodynamic parameters (ΔG, ΔH, and ΔS) indicatedthat entropy-dependent hydrophobic and endothermic interactions played a major role inthe reaction between cNDIs and CT-DNA. Stopped-flow analysis showed that 2 slowlydissociate from GC base pairs. The salt ion effect analysis showed that upon theincreasing salt concentration reduced cNDIs binding with dsDNA. Compound 1 showedmuch slower dissociation, a higher binding selectivity and a more entropically favorableinteraction to dsDNA than 2 because of its longer linker chain.The binding interactions between G-quadruplex DNA structure and cNDIsshowed the affinities in the range of 106?107 M?1 orders with a 2:1 stoichiometry.Compound 2 showed highest binding affinities to human telomere a-core G-quadruplexDNA with 270 times selectivity than dsDNA. The CD spectra of G-quadruplex DNAchanged upon the addition of cNDIs suggesting the end staking interaction of cNDIs onG-tetrad plane. The thermal melting studies indicated that 2 stabilized to G-quadruplexesDNA with preference of human telomeric a-core TT. The FRET melting assay alsoshowed that 2 highly stabilized with F21T which is an ancestor of human telomere G-quadruplex DNA. Compound 2 revealed an effective inhibitor against telomerase activitywith an IC50 value of 0.9 μM.Briefly, the finding of this doctoral thesis will contribute to generate new idea todesign and development suitable DNA binding ligands. The interesting data in the chapter4 indicated that 2 is the suitable candidate drug target to G-quadruplexes DNA, it deservesfor further investigation with cancer cell line.
九州工業大学博士学位論文 学位記番号:工博甲第402号 学位授与年月日:平成27年9月25日
I: Introduction and Background|II: Synthesis of cyclic naphthalene diimide 1, 2 and 3|III: Thermodynamics and Kinetic Studies in the Binding Interaction of Cyclic Naphthalene Diimide Derivatives with Double Stranded DNAs|IV: A Selective G-Quadruplex DNA-Stabilizing Ligand Based on a Cyclic Naphthalene Diimide Derivative|V: Conclusion and perspective