UNS: Biomolecular Engineering of siRNAs

1510895沃尔顿，斯蒂芬·P. RNA干扰（RNAi）是一种天然的细胞途径，可用于减少靶蛋白的表达。这种途径可以被称为短干扰RNA（siRNA）的双链RNA激活。所提出的工作的具体目的是：i）定义siRNA的序列和结构特征，其导致RNAi途径蛋白对siRNA的适当加工; ii）表征影响RNAi途径蛋白对siRNA的结合的siRNA特征;以及iii）确定含siRNA的复合物的细胞摄取途径，其使递送的siRNA的功能最大化。由于RNAi的潜在特异性及其对几乎任何蛋白质靶点的适用性，siRNA作为下一代生物分子治疗剂获得了相当大的关注。RNA干扰有望成为操纵生物过程和开发新一类治疗剂的工具。RNAi由短的干扰RNA（siRNA）启动，并导致靶蛋白的敲低。为了实现敲低，外源siRNA必须被递送到靶细胞的细胞质中并与RNAi途径蛋白有效地相互作用。然而，siRNA递送仍然是一个重大挑战。为了实现沉默，siRNA必须首先通过允许它们活跃的机制进入靶细胞。目前，导致siRNA功能的摄取途径知之甚少。拟议的工作旨在确定管理siRNA设计的规则以及用于将它们传递到细胞的载体。使用体外和细胞系统，将研究具有独特特征的siRNA和递送载体，以确定哪种特征组合导致最高的siRNA活性。所提出的工作的具体目标是i）使用体外系统定义与功能不对称相关的siRNA的序列和结构特征; ii）使用简并siRNA和平行RNA测序表征影响siRNA与RNAi途径蛋白的体外结合相互作用的序列特征;和iii）确定siRNA的细胞摄取途径，该奖项由CBET部门的生物技术和生物化学工程项目授予，由分子和细胞生物学系的系统和合成生物学项目共同资助。

1510895Walton, Stephen P. RNA interference (RNAi) is a natural cellular pathway that can be used to reduce the expression of a targeted protein. This pathway can be activated by double-stranded RNAs called short, interfering RNAs (siRNAs). The specific aims of the proposed work are to: i) define the sequence and structural features of siRNAs that result in the proper processing of siRNAs by the RNAi pathway proteins; ii) characterize the siRNA features that affect binding of the siRNAs by RNAi pathway proteins; and iii) determine the cellular uptake pathways for siRNA-containing complexes that maximize function of the delivered siRNAs. Due to the potential specificity of RNAi and its applicability against nearly any protein target, siRNAs have garnered considerable attention as the next generation of biomolecular therapeutics.RNA interference holds promise as a tool for manipulation of biological processes and for the development of a new class of therapeutics. RNAi is initiated by short, interfering RNAs (siRNAs) and results in the knockdown of a targeted protein. To achieve knockdown, exogenous siRNAs must be delivered to the cytoplasms of targeted cells and interact productively with the RNAi pathway proteins. However, siRNA delivery remains a significant challenge. To achieve silencing, siRNAs must first enter the targeted cells by a mechanism that allows them to be active. Currently, the uptake pathways that result in siRNA function are poorly understood. The proposed work seeks to identify the rules that govern the design of siRNAs and the vehicles used to deliver them to cells. Using in vitro and cellular systems, siRNAs and delivery vehicles with unique characteristics will be studied to determine which combination of features results in the highest siRNA activity. The specific aims of the proposed work are to i) define the sequence and structural features of siRNAs that are associated with functional asymmetry using an in vitro system; ii) characterize the sequence features that influence in vitro binding interactions of siRNAs with RNAi pathway proteins using degenerate siRNAs and parallel RNA sequencing; and iii) determine the cellular uptake pathways for siRNA-containing complexes that maximize silencing efficiency of the delivered siRNAs.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.