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

项目总结/摘要 我们在此提出了一种方法来鉴定基于合成肽的结合剂， RNA中的基序。选择性靶向这些生物学上重要的基序的试剂将是变革性的 用于研究和调节RNA控制的生物学的治疗诊断工具。我们研究的长期目标是 计划是开发序列和上下文选择性试剂，用于靶向任何此类非典型的 lncRNA结构基序。本申请的目的是合成一个小的肽衍生的文库， 试剂并定量地排列它们在与确定的RNA结构的非规范杂交中的能力， 在体外和细胞培养中使用新的和稳健的功能筛选方法。与引人注目的 随着合成生物学在编码界面的进展，非规范靶向仍在开发中。我们 假设这一领域的进展受到以下几个方面的限制：1）先前的重点是针对沃森-克里克 (WC)配对的碱基而不是非典型的碱基对; 2）过度强调插入驱动的结合; 3） 缺乏对RNA靶向试剂中二级结构的探索; 4）缺乏统一的功能测定， 严格评估约束力。我们进一步假设，合成的结合解决方案存在于每一个 如果能找到这些解决方案，那么就可以编程非规范杂交 以与双链体杂交程序相同的方式。这样的进步将使精确的审讯成为可能 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， 合成碱基在靶向选择非典型对，以及初步数据表明，可调和 通过主链和碱基修饰，具有广泛的结合选择性。我们将测试我们的中心假设， 通过以下三个具体目标实现本申请的总体目标：1）合成和 通过碱基三联体形成靶向非典型位点的bPNA的评价; 2）酶促和功能性 bPNA-RNA靶向功效的测定; 3）体外和细胞内bPNA靶向和选择性 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.