Directed Libraries of Self-Circularizing Antisense RNA

描述(由申请人提供)：我们提出了一种基于自循环反义RNA文库的优化基因特异性抑制剂选择的新方法，称为Lassos。Lassos与目标RNA分子杂交并与其形成拓扑连接(通过环化)，产生与目前可用的反义药物相比具有更好的稳定性和序列特异性的复合体。这项建议解决了所有反义方法中的一个主要挑战，即在目标RNA上找到最“敏感”的序列。我们的方法允许同时选择可访问的目标位置和Lassos的最佳循环能力。该项目包括开发一种新的方法来制备基因特异性(定向)文库并将它们整合到套索结构中。 这些定向文库的复杂性和毒性比完全随机文库低得多，这使得它们特别适用于基于细胞的选择。一种新的体外选择方案将被用来鉴定与靶标结合和循环的套索物种，具有快速动力学、高亲和力和序列特异性。该项目的第二阶段将专注于将第一阶段开发的方案应用于细胞选择，以确定哪些Lassos最能特异性地抑制感兴趣基因的表达。通过这种方式，Lassos将以自动和直接的方式针对蜂窝环境中的功能进行优化。它们应该找到不同的应用，包括抑制疾病相关基因的表达，功能基因组学，以及靶点验证。

DESCRIPTION (provided by applicant): We propose a new approach for the selection of optimized gene-specific inhibitors based on libraries of self-circularizing antisense RNAs called Lassos. Lassos hybridize to and become topologically linked (throughcircularization) with target RNA molecules, creating complexes having superior stability and sequence specificitycompared to currently available antisense agents. This proposal addresses a major challenge in all antisense approaches, finding the most "sensitive" sequences on target RNAs. Our approach allows simultaneous selection of both accessible target sites and optimal circularizing ability for Lassos. The project includes the development of a new method for preparing gene-specific (directed)libraries and incorporating them into the Lasso structure. These directed libraries are of much lower complexity and toxicity than fully random libraries, making them especially useful for cell-based selections. A novel in vitro selection scheme will be used to identify Lasso species that bind and circularize around the target with fast kinetics, high affinity and sequence specificity. Phase II of the project will focus on applying the scheme developed in Phase I to selection in cells, to identify those Lassos that are best able to specifically inhibitexpression of a gene of interest. In this way, Lassos will be optimized for function in the cellular environment in an automatic and straightforward manner. They should find diverse applications, includinginhibitingthe expression of disease-related genes, functional genomics, and target validation.