MYC EXPRESSION BY TRIPLEX FORMATION

三链结构的设计及其生物活性研究进展 形成寡核苷酸(TFOS)表明TFOS可能是 发展成为一种基因特异性试剂(Moffat，A.(1991)) 科学252：1374)。总体目标：本提案的目标是 使用c-myc原癌基因作为模型系统进行开发 TFOS作为细胞抗启动子化合物的应用。我们也是 有兴趣探索转化的表型是否在 Burkitt淋巴瘤可能是由c-myc P1过高引起的 启动子的用法。启动子靶点选择：我们已经选择了两个位点 作为最高优先目标：P1站点4，-153/-115 P1起始点和P2起始点1，距离P2起始点-76/-51。 每一项都是配置项中转录启动所必需的，并且每一项都是 几个调节蛋白的结合部位。结构和 生化研究：化学足迹和谱带移位分析 将用于定义稳定性和低分辨率结构 P1和p2c-myc启动子内的TFO复合体。 TFO与c-myc转录因子的直接竞争 结合将在体外通过条带移位方法进行评估。TFO 介导的转录抑制将在体外通过一种 核糖核酸酶保护实验，采用纯化的c-myc因子。蜂窝 摄取和结合研究：摄取速率参数、TFO稳定性 TFOS的核分区将在细胞中进行评估，使用 放射化学方法。重点将放在定义 胆固醇或多聚赖氨酸末端修饰对上述指标的影响 参数。TFOS与其核内DNA靶位的结合 将通过DNaseI保护方法进行评估，通过体内 足迹和高效TFO交联剂的使用 以溴乙酸酯、补骨脂素和叠氮交联物为基础的结合物 化学反应。细胞抗启动子活性：C-myc抗启动子 细胞中的活性将通过一种变种在信使核糖核酸水平上进行评估。 RNaseI保护试验和聚合酶链式反应方法。这些细胞系 将研究HeLa、Burkitt淋巴瘤和MCF7。

Recent progress in the design and biological activity of triplex forming oligonucleotides (TFOs) has suggested that TFOs might be developed into a type of gene specific reagent (Moffat, A. (1991) Science 252:1374). Overall goals: The goal of this proposal is to use the c-myc protooncogene as a model system in which to develop the use of TFOs as cellular anti-promoter compounds. We are also interested in exploring whether the transformed phenotype in Burkitt's lymphoma may result from inappropriately high c-myc P1 promoter usage. Promoter target selection:We have chosen two sites as highest priority targets: P1 Site 4, -153/-115 with respect to the P1 start site and P2 Site 1, -76/-51 from the P2 start site. Each is required in cis for transcription initiation and each is the binding site for several regulatory prothins. Structural and biochemical studies: Chemical footprinting and band shift analysis will be used to define the stability and low resolution structure of TFO complexes with sites within the P1 and P2 c-myc promoter. Direct competition between TFO and c-myc transcription factor binding will be assessed in vitro by a band shift method. TFO mediated transcription inhibition will be assessed in vitro, by a RNase protection assay, employing purified c-myc factors. Cellular uptake and binding studies: Uptake rate parameters, TFO stability and nuclear partitioning of TFOs will be assessed in cells using radiochemical methods. Emphasis will be given to defining the effect of cholesterol or polylysine end-modification on those parameters. Binding of TFOs to their DNA target site in the nucleus will be assessed by a DNaseI protection method, by in vivo footprinting and by the use of high efficiency TFO-crosslinker conjugates, based upon bromoacetate, psoralen and azido crosslinking chemistry. Cellular anti-promoter activity: c-myc anti-promoter activity will be assessed in cells at the mRNA level by a variant of the RNaseI protection assay and by a PCR method. The cell lines to be studied will be HeLa, Burkitt lymphoma and MCF7.