Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
NMR based Structure and Stabilization of G quadruplex d-(TTGGGGT)4 due to binding with an alkaloid Coralyne
G-quadruplex DNA are distributed in human genome particularly in telomeric DNA, oncogenes, gene promoters, transcriptome and correlate with gene expression level by interacting with array of proteins. The general consensus is that G-quadruplex binders which stabilize G-quadruplex structure, interfere with DNA damage response activation, oncogene expression and genomic stability and hence possess potential to act as regulatory elements of different processes. The structure-based design approaches based on molecular interactions of ligands, specific with G-quadruplex, thus find potential therapeutic applications. Using Telomerase Repeat Amplification Protocol assay, we find that Coralyne, an alkaloid (picture on left), inhibits telomerase enzyme with EC50 = 10 µM The circular dichroism spectra obtained on titration of G-quadruplex sequence d-(TTGGGGT)4 (dashed curve in picture on right) with coralyne indicates change in 260 and 246 nm DNA bands as well as appearance of induced CD bands at 310 nm (continuous curves at different coralyne to DNA molar ratios in picture on right). The proton NMR spectra obtained on successive addition of coralyne to d-(TTGGGGT)4 show significant shifts in base and NH protons. The G3NH and G6NH broaden and shift by 0.28-0.56 ppm upfield which is much greater than the corresponding changes in G4NH and G5NH protons. Slow exchange between bound and free NH protons is observed at 278 K. The G3NH and G6NH protons disappearing at 55 °C persist up to 65 °C at drug to DNA added molar ratio of 1.0 and get further stabilized up to 80 °C at molar ratio of 2.0. The protons attached directly to the aromatic ring as well as OCH3 protons of coralyne exhibit upfield shifts in the range 0.09-0.46 ppm on binding to the G-quadruplex DNA. Short inter proton intermolecular connectivities are observed between coralyne protons and TCH3 and GH8 base protons. The restrained molecular dynamics simulations performed using ∼12 Nuclear Overhauser enhancement connectivities (NOEs) gives conformation of the coralyne bound to parallel form of G-quadruplex. The observed specific interactions may be attributed to enhanced thermal stabilization of DNA quadruplex which is capable of inhibition of telomerase enzyme.
Department of Biotechnology