G-quadruples Topology-selective Probes

有了这个奖项，化学系的生命过程化学项目正在资助博士。来自密西西比州立大学的Edwin Lewis和来自马里兰大学的Herman Sintim开发了新的化学探针，用于在复杂的生物环境中研究g -四重体生物学。g -四联体是由DNA或RNA中富含鸟嘌呤的短序列形成的独特折叠的非螺旋结构，涉及许多重要生命过程的调节，从基因表达和mRNA剪接，到染色体维持，细胞增殖和病毒生命周期。与双链螺旋DNA相比，开发对g -四重体DNA具有很高亲和力，并能够选择特定的g -四重体折叠拓扑结构的化学探针是生物化学和分子生物学研究的新方向。新型化学探针的设计，它们的合成以及探针/ g -四复体复合物在体外和体内的生物物理特性将为研究生和高级本科生提供机会，以获得新化合物合成和使用光谱，微热量，核磁共振，质谱和分子生物学方法的各种专业技能。本项目旨在解决g -四重体生物学体内研究的核心问题；缺乏选择性探针来区分：a)丰富的双链核酸和少得多的g -四链核酸；b) DNA g -四重体和RNA g -四重体；c)在几个g -四重结构或构象之间。除了这些尚未解决的选择性问题外，迄今为止开发的大多数g -四重体配体仅具有中等的结合亲和力，只有少数g -四重体具有纳摩尔Kd值。主要研究人员最近发现了一个令人惊讶的发现，一种用于兽医学的药物，据信通过与Kd约为1微摩尔的富含at的双链的小凹槽结合而起作用，与Kds约为1纳摩尔的g -四联体结合，这是迄今为止报道的g -四联体化学探针的最强亲和力。基于这一发现，本项目将进行基于DMZ支架的新型g -四联体化学探针的开发。将使用各种生物物理工具来研究新化学探针对不同g -四plex拓扑（例如平行与反平行，小环与大环g -四plex）和类型（DNA与RNA）的选择性。核酸结合蛋白与各种探针稳定的g -四重结构的相互作用也将在本项目中进行研究。本研究提供的信息有望为研究g -四重体核酸结构在许多细胞过程中的作用提供工具。

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.