Directed Libraries of Self-Circularizing Antisense RNA

描述（由申请人提供）：我们提出了一种基于称为Lassos的自环化反义RNA文库来选择优化的基因特异性抑制剂的新方法。Lasso与靶RNA分子杂交并拓扑连接（通过环化），产生与目前可用的反义试剂相比具有上级稳定性和序列特异性的复合物。该提议解决了所有反义方法中的一个主要挑战，即找到靶RNA上最“敏感”的序列。我们的方法允许同时选择两个可访问的目标网站和最佳的环化能力的Lassos。该项目包括开发一种制备基因特异性（定向）文库并将其纳入Lasso结构的新方法。 这些定向文库比完全随机文库具有低得多的复杂性和毒性，使得它们对于基于细胞的选择特别有用。将使用一种新的体外选择方案来鉴定Lasso种类，其以快速动力学、高亲和力和序列特异性结合并环化靶标。该项目的第二阶段将侧重于将第一阶段开发的方案应用于细胞选择，以鉴定那些最能特异性抑制感兴趣基因表达的Lassos。通过这种方式，Lassos将以自动和直接的方式针对细胞环境中的功能进行优化。他们应该找到不同的应用，包括检测疾病相关基因的表达，功能基因组学和目标验证。

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.