RNA Aptamers to Green Fluorescent Protein for Cell Imaging

描述（由申请人提供）：这项探索性资助旨在开发一种新颖且有前途的细胞内成像系统。我们已经鉴定了结合并显著降低绿色荧光蛋白的荧光的RNA适体。所鉴定的适体结构以高亲和力结合GFP、eGFP、YFP和CFP，但选择性地抑制GFP和eGFP的荧光发射（KD = 14 nM），主要通过摩尔消光系数的降低。在这里，我们建议利用荧光猝灭RNA适体作为一个灵活的，遗传编码的分子检测系统。我们将优化基因构建体的核和细胞质RNA适体的表达（目的1）。荧光蛋白结合结构将通过与第二检测器结构域连接而用作二价RNA的变构调节的报告组分（Aim 2）;靶分子与RNA适体内的检测器结构域的结合将导致荧光猝灭RNA适体与GFP的解结合并诱导稳健的荧光信号。这样的系统可以提供一种新的方法，在体外和体内细胞成像，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。光学成像和遗传规格的最新进展已经彻底改变了我们对复杂生理过程的理解，例如发育和对疾病的反应。该提案旨在开发一种灵活且广泛适用的方法，用于构建遗传编码的光学传感器，该传感器将报告活细胞中特定分子的浓度，位置和结构，从而显着扩展用于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.