ANTISENSE AND TRIPLEX DNA FORMATION IN CELLS BY ESR

通过 ESR 观察细胞中反义 DNA 和三链体 DNA 的形成

• 批准号: 2605383
• 负责人: Peter M. Gannett
• 金额: $ 10.94万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2605383
• 关键词: DNA; antisense nucleic acid; cell line; electron spin resonance spectroscopy; gel electrophoresis; molecular biology; nucleic acid biosynthesis; oligonucleotides; simian virus 40; tissue /cell culture; triple helix

DESCRIPTION (Adapted from applicant's abstract): Antisense and triplex DNA (tx DNA) based pharmaceuticals offer an exciting approach to treat gene-based diseases with the potential for high selectivity and low toxicity. The molecular basis for these technologies offers new tools in molecular biology. At the molecular level, antisense and tx DNA agents act by binding to mRNA and DNA, respectively, and thereby inhibit gene expression. These agents have been extensively studied by spectroscopic methods in relatively simple chemical systems. However, the spectroscopic methods currently used have not been applied to more complicated and biologically relevant systems due to current sensitivity and resolution limitations. Therefore, more indirect and inconclusive methods have been used to study the activity of antisense and tx DNA oligonucleotides (ONs) in cellular systems, severely limiting the current understanding of these agents. The development of antisense and tx DNA agents would be facilitated if a more robust spectroscopic method were available for their study in cellular systems. A method that has been used to study biochemical processes in cellular systems is the spin probe labeling method. This method utilizes a substrate suitable labeled with a spin probe that can be monitored by ESR and is quite sensitive and selective. Here the investigators propose to extend the spin probe method by applying it to the study of antisense and tx DNA based pharmaceutical agents. ONs will be prepared that are spin probe labeled on a base or on a DNA intercalator covalently bonded to the end of the ON. The modified ONs will be compared with the corresponding unmodified ONs by thermal denaturation and gel electrophoresis methods. The ESR signatures unique to these spin probe labeled ONs in double stranded DNA (ds DNA) or tx DNAs (base or intercalator spin labeled) will be determined. The ONs will also be used to determine association constants for tx DNA formation. There are few methods available for measuring association constant formation for tx DNA and this is an important quantity to measure since it reflects the effectiveness of binding to the target. Finally, the investigators will examine the formation of tx DNA in a cellular system. CV-1 cells infected with SV40 virus will be treated with an ON designed to bind to the SV40 DNA, forming a tx DNA. The process will be monitored by ESR for tx DNA formation, assayed for their cytopathic effects and the ESR data correlated with the cytopathic effect data. The development of the ESR spin probe technique for studying antisense and tx DNA will significantly aid the development of these agents by providing a tool to monitor their binding to their cellular targets. Moreover, these agents may also be used in the future to study other aspects of these agents including cellular uptake and concentration in the nucleus. Likewise, by extension of the studies proposed here, these agents may be useful for studying other novel DNA structures in cells such as telomeric DNA.

描述（改编自申请人摘要）：反义和三链体DNA (tx基于DNA）的药物提供了一种令人兴奋的治疗方法 基于基因的疾病，具有高选择性和低选择性的潜力， 毒性 这些技术的分子基础提供了新的工具， 分子生物学 在分子水平上，反义和反义DNA试剂起作用， 通过分别与mRNA和DNA结合，从而抑制基因 表情 这些代理人已被广泛研究的光谱 在相对简单的化学体系中。 然而，光谱 目前使用的方法还没有应用于更复杂的， 由于电流灵敏度和分辨率，生物相关系统 局限性。 因此，更多的间接和不确定的方法已经被 用于研究反义和tx DNA寡核苷酸（ON）在 蜂窝系统，严重限制了目前对这些的理解 剂. 反义和tx DNA试剂的发展将是促进，如果一个 更稳健光谱方法可用于它们在细胞中的研究 系统. 一种用于研究生物化学过程的方法， 细胞系统是自旋探针标记方法。 该方法利用了 适合用可通过ESR监测的自旋探针标记的基底 并且非常敏感和有选择性。 在这里，研究人员建议， 将自旋探针法推广到反义核酸和TX的研究 基于DNA的药剂。 将制备自旋探针ON 标记在碱基上或共价键合到末端的DNA嵌入剂上 将修改的ON与对应的未修改的ON进行比较。 通过热变性和凝胶电泳方法测定ONs。 的ESR 这些自旋探针标记的双链DNA（ds）中的ON所特有的特征 DNA）或tx DNA（碱基或嵌入剂自旋标记的）。 的 ON还将用于确定tx DNA的缔合常数 阵 测量关联性的方法很少 这是一个重要的测量量 因为它反映了绑定目标的有效性。 最后 研究人员将在细胞系统中检查tx DNA的形成。 用设计为 与SV40 DNA结合，形成TX DNA。 该过程将由以下人员监测： 用于tx DNA形成的ESR，测定其细胞病变效应和ESR 与细胞病变效应数据相关的数据。 ESR自旋探针技术在反义核酸研究中的应用 tx DNA将通过提供一种 工具来监测它们与细胞靶点的结合。 而且这些 未来还可能使用这些试剂来研究这些试剂的其他方面 包括细胞摄取和细胞核中的浓缩。 同样，由 扩展这里提出的研究，这些代理人可能是有用的， 研究细胞中其他新的DNA结构，如端粒DNA。