RNA Aptamers to Green Fluorescent Protein for Cell Imaging

描述(由申请人提供)：这项探索性拨款旨在开发一种新的和有前途的细胞内成像系统。我们已经确定了与绿色荧光蛋白结合并显著降低其荧光的RNA适配子。鉴定的适体结构以高亲和力与GFP、EGFP、YFP和CFP结合，但选择性地抑制GFP和EGFP的荧光发射(Kd=14 nM)，主要是通过降低摩尔消光系数来实现的。在这里，我们建议利用荧光猝灭RNA适配子作为一种灵活的、可遗传编码的分子检测系统。我们将优化核质RNA适配子表达的遗传结构(目标1)。荧光蛋白结合结构将通过连接到第二检测器域(AIM 2)而用作二价RNA的变构调节的报告成分；靶分子与RNA适配子内的检测器结构域的结合将导致荧光猝灭RNA适配子从GFP上解离并诱导出强大的荧光信号。这样的系统可以为体外和体内细胞成像提供一种新的方法，也可以适用于快速的生物危害检测。细胞内和细胞间的生化信号是正常和异常功能的基础。光学成像和基因规范的最新进展彻底改变了我们对复杂生理过程的理解，如发育和对疾病的反应。这项提议旨在开发一种灵活且广泛适用的方法来构建基因编码的光学传感器，该传感器将报告活细胞中特定分子的浓度、位置和结构，从而显著扩展理解复杂细胞反应的工具。

DESCRIPTION (provided by applicant): This exploratory grant seeks to develop a novel and promising system for intracellular imaging. We have identified RNA aptamers that bind to and markedly reduce the fluorescence of Green Fluorescent Proteins. The aptamer structures identified bind to GFP, eGFP, YFP, and CFP with high affinity, but selectively inhibit the fluorescence emission of GFP and eGFP (KD = 14 nM), largely through a decrease in the molar extinction coefficient. Here we propose to exploit fluorescence quenching RNA aptamers as a flexible, genetically encodable molecular detection system. We will optimize genetic constructs for the expression of nuclear and cytoplasmic RNA aptamers (Aim 1). The fluorescent protein binding structure will be utilized as an allosterically regulated reporter component of a bivalent RNA by linkage to a second detector domain (Aim 2); binding of a target molecule to the detector domain within the RNA aptamer will resulting in unbinding of the fluorescence quenching RNA aptamer from GFP and the induction of a robust fluorescent signal. Such a system could provide a novel approach to in vitro and in vivo cellular imaging, and could also be adapted to rapid biohazard detection. Biochemical signaling within cells and between cells underlies normal and abnormal function. Recent advances in optical imaging and genetic specification have revolutionized our understanding of complex physiological processes such as development and the response to disease. This proposal seeks to develop a flexible and broadly applicable method for the construction of genetically encoded optical sensors that will report on the concentration, location, and structure of specific molecules in live cells, thereby markedly expanding the tools for understanding complex cellular responses.