UNS: Biomolecular Engineering of siRNAs

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。这一途径可以被称为短干扰RNA(SiRNAs)的双链RNA激活。这项拟议工作的具体目标是：i)确定siRNAs的序列和结构特征，从而导致RNAi途径蛋白对siRNAs的适当处理；ii)表征影响RNAi途径蛋白与siRNAs结合的siRNA特征；以及iii)确定最大限度地发挥所传递的siRNAs功能的含有siRNA的复合体的细胞摄取途径。由于RNAi的潜在特异性和对几乎任何蛋白质靶点的适用性，siRNAs作为下一代生物分子疗法受到了极大的关注，RNA干扰有望成为操纵生物过程和开发一类新的治疗学的工具。RNAi是由短的干扰RNAs(SiRNAs)启动的，并导致目标蛋白的击倒。为了实现击倒，外源siRNAs必须被输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的传递仍然是一个巨大的挑战。为了实现沉默，siRNA必须首先通过一种允许它们激活的机制进入靶细胞。目前，导致siRNA功能的摄取途径还知之甚少。这项拟议的工作旨在确定管理siRNA设计的规则和用于将它们运送到细胞的工具。利用体外和细胞系统，将研究具有独特特征的siRNA和递送载体，以确定哪种特征的组合可以产生最高的siRNA活性。这项拟议工作的具体目标是：i)使用体外系统确定与功能不对称相关的siRNAs的序列和结构特征；ii)使用简并的siRNAs和并行RNA测序来表征影响siRNAs与RNAi途径蛋白体外结合作用的序列特征；以及iii)确定含有siRNA的复合体的细胞摄取途径，从而最大限度地提高交付的siRNAs的沉默效率。

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.