G-quadruples Topology-selective Probes

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With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Drs. Edwin Lewis from Mississippi State University and Herman Sintim from the University of Maryland to develop new chemical probes for the study of G-quadruplex biology in complex biological environments. G-quadruplexes, uniquely folded non-helical structures formed by short guanine rich sequences in DNA or RNA, have been implicated in the regulation of a number of important life processes, from gene expression and mRNA splicing, to chromosome maintenance, cell proliferation, and viral life cycles. The development of chemical probes having very high affinity for G-quadruplex DNA over double stranded helical DNA, and having the ability to select for specific G-quadruplex folding topologies represents a new direction in biochemistry and molecular biology. The design of the new chemical probes, their synthesis and the biophysical characterization of the probe/G-quadruplex complexes in vitro and in vivo will provide opportunities for graduate students and advanced undergraduates to acquire a diverse set of specialized skills in new compound synthesis and in the use of spectroscopic, microcalorimetric, NMR, mass spectrometry, and molecular biology methods. This project is designed to address the central issue in the in vivo study of G-quadruplex biology; the lack of selective probes that can discriminate between: a) abundant duplex nucleic acids and much less abundant G-quadruplexes; b) DNA G-quadruplexes and RNA G-quadruplexes; c) and among several G-quadruplex topologies or conformers. In addition to these unresolved selectivity issues, most G-quadruplex ligands that have been developed to date have only moderate binding affinities with only a handful of G-quadruplex ligands exhibiting nanomolar Kd values. The principal investigators, recently made a surprising discovery that diminazene (DMZ), a drug that is used in veterinary medicine and believed to act via binding to the minor groove of AT-rich duplexes with a Kd of about 1 micromolar, binds to selected G-quadruplexes with Kds of about 1 nanomolar - the strongest affinity reported so far for a G-quadruplex chemical probe. Based on this discovery, this project will undertake the development of new G-quadruplex chemical probes based on the DMZ scaffold. Various biophysical tools will be employed to study the selectivity of the new chemical probes for different G-quadruplex topologies (e.g. parallel vs. antiparallel, and small loop vs. large loop G-quadruplexes) and types (DNA vs. RNA). The interactions of nucleic acid binding proteins with various probe-stabilized G-quadruplex structures will also be investigated in this project. Information developed in this study is expected to provide tools to study the role of G-quadruplex nucleic acid structures in a number of cellular processes.