ANTISENSE AND TRIPLEX DNA FORMATION IN CELLS BY ESR

描述(改编自申请人的摘要)：反义和三链DNA (TX DNA)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TX DNA试剂起作用 通过分别与mRNA和DNA结合，从而抑制基因 表情。这些试剂已经通过光谱学进行了广泛的研究 方法在相对简单的化学体系中。然而，光谱 目前使用的方法尚未应用于更复杂和 基于电流灵敏度和分辨率的生物相关系统 限制。因此，出现了更多间接和不确定的方法。 用来研究反义和Tx DNA寡核苷酸(ONS)在小鼠体内的活性 蜂窝系统，严重限制了目前对这些系统的理解 探员们。 反义和TX DNA试剂的发展将得到促进 更可靠的光谱方法可用于细胞内的研究 系统。一种已被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。此方法利用 适合用可由ESR监测的自旋探针标记的底物 而且非常敏感和有选择性。在这里，调查人员提议 自旋探针法在反义和毒素研究中的应用 基于DNA的药物制剂。将准备自旋探测器的ONS 标记在碱基上或共价连接到末端的DNA嵌入物上 开机了。将修改的ONS与相应的未修改的ONS进行比较 采用热变性和凝胶电泳法。ESR 这些自旋探针在双链DNA(DS)中标记ONS的独特特征 DNA)或TX DNA(碱基或嵌入体自旋标记)将被确定。这个 ONS还将用于确定TX DNA的结合常数 队形。测量关联性的方法很少 TX DNA的恒定构型，这是一个需要测量的重要量 因为它反映了绑定到目标的有效性。最后， 研究人员将检查TX DNA在细胞系统中的形成。 感染SV40病毒的CV-1细胞将被设计为 与SV40 DNA结合，形成TX DNA。这一过程将由以下人员监督 TX DNA形成的ESR，分析它们的细胞病变效应和ESR 与细胞病变效应数据相关的数据。 反义和反义ESR自旋探针技术的研究进展 TX DNA将通过提供一种 工具来监测它们与细胞靶标的结合。此外，这些 这些试剂将来也可能被用来研究这些药物的其他方面。 包括细胞核内的细胞摄取和浓缩。同样，通过 扩展这里提出的研究，这些试剂可能对 研究细胞中的其他新DNA结构，如端粒DNA。

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.