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.
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