Synthetic strategies for non-canonical hybridization to structural motifs in RNA

项目摘要/摘要 我们在这里提出了一种方法来识别基于合成肽的结合到非正则结构 RNA中的基序。选择性地针对这些生物重要基序的试剂将是变革性的 研究和调节RNA控制的生物学的声学工具。我们研究的长期目标是 该计划是开发序列和上下文选择性试剂，用于靶向任何这样的非规范 IncRNA中的结构基序。本申请目的是合成一个小的多肽衍生的文库 并对它们在与已定义的RNA结构的非正则杂交中的能力进行定量排序， 在体外和细胞培养中使用新的和强大的功能筛选方法。与罢工形成对比的是 合成生物学在编码界面、非规范靶向方面的进展仍在开发中。我们 假设这一领域的进展在几个方面受到限制：1)优先关注沃森-克里克 (WC)成对碱基而不是非正则对；2)过分强调插层驱动的结合；3) 缺乏对RNA靶向试剂二级结构的探索；4)缺乏统一的功能分析方法 严格评估绑定情况。我们进一步假设，合成绑定解决方案存在于每个 非规范主题；如果可以找到这些解决方案，那么非规范杂交就可以被编程 以同样的方式，双链杂交被编程。这样的进步将使精确审讯成为可能 用新的化学工具研究核酸生物学。先前研究的严谨性在于已知的效率 以选定的非正则对为目标的合成基础，以及展示可调和 通过主链和碱基修饰的扩张性结合选择性。我们将测试我们的中心假设，并 通过以下三个具体目标实现本申请的总体目标：1)综合和 通过碱基三重构象评价靶向非正则位点的bPNA2)酶活性和功能性 BPNA-RNA靶向性的测定；3)体外和细胞内的bPNA靶向性和选择性 天然RNA中的结构基序。

PROJECT SUMMARY/ABSTRACT We propose herein a methodology to identify synthetic peptide-based binders to non-canonical structural motifs in RNA. Reagents that selectively target these biologically important motifs would be transformative theranostic tools for study and modulation of RNA-governed biology. The long-term goal of our research program is to develop sequence and context-selective reagents for targeting of any such non-canonical structural motif in lncRNAs. The objective of this application is to synthesize a small library of peptide-derived reagents and quantitatively rank their competence in non-canonical hybridization to defined RNA structures, using novel and robust functional screening methods in vitro and in cell culture. In contrast to the striking progress of synthetic biology at the coding interface, non-canonical targeting remains in development. We hypothesize that progress in this area is limited in a number of ways: 1) a prior focus on targeting Watson-Crick (WC) paired bases rather than non-canonical pairs; 2) an over-emphasis on intercalation-driven binding; 3) lack of exploration of secondary structure in RNA targeting reagents; 4) lack of a unified functional assay to rigorously evaluate binding. We further hypothesize that synthetic binding solutions exist for every non-canonical motif; if these solutions could be found, then non-canonical hybridization could be programmed in the same way that duplex hybridization is programmed. Such an advance would enable precise interrogation of nucleic acid biology with novel chemical tools. The rigor of the prior research lies in the known efficiency of synthetic bases in targeting select non-canonical pairs, as well as preliminary data demonstrating tunable and expansive binding selectivity via backbone and base modification. We will test our central hypothesis and accomplish the overall objective of this application via the following three specific aims: 1) Synthesis and evaluation of bPNAs targeting non-canonical sites via base-triple formation; 2) Enzymatic and functional assays for bPNA-RNA targeting efficacy; 3) In vitro and intracellular bPNA targeting and selectivity for structural motifs in native RNAs.