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

描述（由申请人提供）：富含鸟嘌呤的DNA和RNA序列可以折叠成由堆叠的鸟嘌呤四联体组成的结构。这些四链体结构被认为在几种生物学背景中发挥重要作用，包括抑制c-MYC癌基因的转录，控制hTERT的前体mRNA的选择性剪接，端粒酶的逆转录酶组分和调节端粒酶的活性。与这些四链体结构结合的分子可以作为生物功能和潜在治疗的探针。 高亲和力PNA探针将被合成并靶向DNA和RNA G-四链体结构。这个建议与标准的反基因和反义方法调节基因表达的区别在于，PNA将具有与其靶同源而不是互补的序列。这种策略是可行的，因为已知富含G的PNA与同源DNA和RNA序列形成杂交四链体。杂交热力学和动力学将测量PNA-DNA和PNA-RNA四链体的形成，使用光谱学和表面等离子体共振实验的组合。这个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。一旦实现这一点，将合成PNA并用于测试与基于生物学相关靶标建模的三个G四链体（2个DNA和1个RNA）的结合。最后，将PNA引入哺乳动物细胞培养物中以研究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.