Targeting Biologically Relevant DNA and RNA with Guanine Quadruplex Forming PNA's

描述(申请人提供)：富含鸟嘌呤的DNA和RNA序列可以折叠成由堆叠的鸟嘌呤四分体组成的结构。这些四链结构被认为在多种生物学环境中发挥重要作用，包括抑制c-myc癌基因的转录，控制hTERT前mRNA的选择性剪接，端粒酶的逆转录酶成分，以及调节端粒酶的活性。结合到这些四链结构上的分子可以作为生物功能和潜在治疗的探针。 将合成高亲和力的PNA探针，并针对DNA和RNAG-四链结构。这一建议与标准的反基因和反义基因表达调控方法的不同之处在于，PNA将具有与其靶标同源而不是互补的序列。这一策略是可行的，因为已知富含G的PNA与同源DNA和RNA序列形成杂交四链。将结合光学光谱和表面等离子体共振实验，测量PNA-DNA和PNA-RNA四链形成的杂交热力学和动力学。该项目的一个重要组成部分将是合成各种PNA设计，以优化亲和力和选择性。一旦实现了这一点，将合成PNA并用于测试与三个G四链(2个DNA和1个RNA)的结合，这些G四链是以生物相关的靶标为模型的。最后，将PNAS引入哺乳动物细胞培养中，研究其对c-myc转录的抑制、hTERT前mRNA剪接的改变和端粒酶活性的抑制。

DESCRIPTION (provided by applicant): Guanine-rich DNA and RNA sequences can fold into structures consisting of stacked guanine tetrads. These quadruplex structures are believed to play important roles in several biological contexts, including repressing transcription of the c-MYC oncogene, controlling alternative splicing of the pre-mRNA for hTERT, the reverse transcriptase component of telomerase and regulating the activity of telomerase. Molecules that bind to these quadruplex structures can serve as probes of biological function and potential therapeutics. High affinity PNA probes will be synthesized and targeted to DNA and RNA G-quadruplex structures. What distinguishes this proposal from standard antigene and antisense approaches to regulating gene expression is the fact that the PNAs will have homologous, rather than complementary, sequences to their targets. This strategy is feasible because G-rich PNAs are known to form hybrid quadruplexes with homologous DNA and RNA sequences. Hybridization thermodynamics and kinetics will be measured for PNA-DNA and PNA-RNA quadruplex formation using a combination of optical spectroscopy and surface plasmon resonance experiments. An important component of this project will be synthesizing various PNA designs will to optimize affinity and selectivity. Once this is achieved, PNAs will be synthesized and used to test binding to three G quadruplexes (2 DNA and 1 RNA) modeled on biologically relevant targets. Finally, PNAs will be introduced into mammalian cell culture to study inhibition of c-MYC transcription, alteration of hTERT pre-mRNA splicing and inhibition of telomerase activity.