MANIPULATION OF C-MYC EXPRESSION BY TRIPLEX FORMATION

三链体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 开发成一种基因特异性试剂（Moffat，A.（1991年） Science 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， TFO作为细胞抗促进剂化合物的用途。 我们也 感兴趣的是探索转化的表型是否在 伯基特淋巴瘤可能由c-myc P1不适当的高水平引起 启动子使用 启动子靶点选择：我们选择了两个位点 作为最高优先级目标：P1站点4， P1起始位点和P2位点1，距P2起始位点-76/-51。 每一个都是转录起始顺式所需的，并且每一个都是转录起始顺式所需的。 几种调节蛋白的结合位点。 结构和 生化研究：化学足迹法和谱带位移分析 将用于定义稳定性和低分辨率结构 的TFO复合物与P1和P2的c-myc启动子内的网站。 TFO与c-myc转录因子的直接竞争 结合将通过带移法在体外评估。 TFO 介导的转录抑制将在体外通过 核糖核酸酶保护试验，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 和TFO的核分配将在细胞中使用 放射化学方法。 重点将放在定义 胆固醇或聚赖氨酸末端修饰对那些 参数 TFO与其在细胞核中的DNA靶位点的结合 将通过DNA酶I保护方法、体内 足迹和使用高效的TFO-交联剂 基于溴乙酸酯、庚烯和叠氮基交联的共轭物 化学. 细胞抗启动子活性：c-myc抗启动子 活性将在细胞中在mRNA水平上通过一种变体 的RNaseI保护测定和通过PCR方法。 的细胞系 将被研究的是HeLa、伯基特淋巴瘤和MCF 7。

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.