Conformation and Multimeric Formation of G-Quadruplexed DNA: Effects of Ligand In

描述（由申请人提供）：端粒酶活性与细胞不朽和肿瘤发生有关。它通过在3'-单链端粒上添加重复的（TTAGGG）n鸟嘌呤丰富序列来负责染色体DNA的延伸。富鸟嘌呤序列可在体外折叠形成g -四联体结构，并可抑制端粒酶，为控制肿瘤发生提供潜在靶点。然而，稳定四重体结构的四重体相互作用剂（QIAs）的发展仍然缓慢，因为四重体在溶液中的多态性构象和沿端粒DNA的潜在四重体多聚。因此，需要对控制DNA四重构象和稳定性的基本化学和物理规则的知识的显著增加，以合理地开发用于化疗应用的qas。本研究的总体目标集中在生理条件下g -四联体DNA的构象和多聚。应用的目标集中在：1)研究不同溶液条件对四重构象稳定性的影响及其对结合模型QIA配体的调制；2)研究多聚四重构象形成的构象驱动力和QIA相互作用的影响。我们开发了一个新的荧光标记的人类端粒序列家族（h(TTAGGG)4），通过使用荧光鸟嘌呤类似物（6-MI）特异性替换单个鸟嘌呤残基。结合cd测量，电泳和UV方法进行表征研究，这些荧光端粒序列可以提供关于人类四联体序列和折叠构象中独特鸟嘌呤位点的局部环境影响的光谱信息。所提供的信息将使我们开始了解单个鸟嘌呤残基的构象异质性在稳定和调节溶液中的四重构象和多聚体构象中的作用，并利用这些信息合理设计具有高四重结合选择性和亲和力的qas。

DESCRIPTION (provided by applicant): Telomerase activity is linked with cellular immortality and tumorigenesis. It is responsible for the elongation of chromosomal DNA through addition of repetitive (TTAGGG)n guanine-rich sequences to the 3'-single- stranded telomeric overhang. The guanine-rich sequences can fold in vitro to form G-quadruplexes structures, and can inhibit telomerase providing a potential target for controlling tumorigenesis. However, development of quadruplex interactive agents (QIAs) that stabilize the quadruplex structure for clinical application remains slow, because of the polymorphic conformation of quadruplexes in solution and potential quadruplex multimerization along the telomeric DNA. A significant increase in knowledge of the fundamental chemical and physical rules that govern DNA quadruplex conformation and stabilization is thus required for rational development of QIAs for chemotherapeutic applications. The overall goal of the proposed research focuses on conformation and multimerization of G-quadruplexed DNA under physiological conditions. The objectives of the application focus on: 1) examining the effects of varying solution conditions on quadruplex conformational stability and its modulation on binding model QIA ligands; and 2) to examine conformational driving forces involved in multimeric quadruplex formation and the effects of QIA interactions. We have developed a novel family of fluorescently labeled human telomeric sequences (h(TTAGGG)4) through site-specific replacement of single guanine residues using a fluorescent guanine analog (6-MI). In combination with CD-measurements, electrophoretic and UV approaches for characterization studies, these fluorescent telomeric sequences can provide spectroscopic information about local environmental effects at unique guanine sites along the human quadruplex sequence and when in the folded conformation. The information provided will allow us to begin to understand the role of conformational heterogeneity of individual guanine residues in stabilizing and modulating the quadruplex conformation and multimeric formation in solution, and to use this information for rational design of QIAs with high quadruplex binding selectivity and affinity. PUBLIC HEALTH RELEVANCE: The focus of this proposal is on sequences of DNA that fold into a four-stranded structure called a quadruplex and can inhibit the activity of a key protein called telomerase, which is linked with cancer. The proposed studies will provide information on what influences shape and organization of the quadruplex particularly in solution. This basic knowledge is critically important as it will allow us to better design Quadruplex Interactive Agent (QIA) therapeutics that bind more effectively and selectively and promote quadruplex folding of the DNA in order to inhibit telomerase and control tumorigenesis.