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

描述(申请人提供)：端粒酶活性与细胞永生和肿瘤发生有关。它通过在3‘-单链端粒悬垂上添加重复的(TTAGGG)n富鸟嘌呤序列来延长染色体DNA。这些富含鸟嘌呤的序列可以在体外折叠形成G-四链结构，并可以抑制端粒酶，为控制肿瘤的发生提供了一个潜在的靶点。然而，由于溶液中四链构象的多态和端粒DNA上潜在的四链多聚体的存在，稳定四链结构用于临床应用的四链相互作用剂(QIA)的开发仍然缓慢。因此，为了合理开发用于化疗的QIA，需要显著增加对支配DNA四链构象和稳定性的基本化学和物理规则的了解。这项研究的总体目标集中在生理条件下G-四链DNA的构象和多聚化。应用的目标集中在：1)检测不同的溶液条件对四链构象稳定性的影响及其对结合模型QIA配体的调节；以及2)检测参与多聚体四链形成的构象驱动力和QIA相互作用的影响。我们开发了一类新的荧光标记的人端粒序列(h(TTAGGG)4)，通过用荧光鸟嘌呤类似物(6-MI)定点替换单个鸟嘌呤残基来实现。结合CD测量、电泳法和UV法进行表征研究，这些荧光端粒序列可以提供关于沿着人类四链序列的独特鸟嘌呤位置以及当处于折叠构象时的局部环境效应的光谱信息。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四链构象和多聚体形成中的作用，并利用这些信息来合理设计具有高四链结合选择性和亲和力的QIA。

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.