RNA Aptamers to Green Fluorescent Protein for Cell Imaging

用于细胞成像的绿色荧光蛋白 RNA 适体

• 批准号: 7318372
• 负责人: Michael I. Kotlikoff
• 金额: $ 23.03万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-05 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7318372
• 关键词: Affinity; Binding; Biochemical; Cell Nucleus; Cells; Complex; Cyclophosphamide/Fluorouracil/Prednisone; Cytosol; Detection; Development; Disease; Equilibrium; Extinction (Psychology); Facility Construction Funding Category; Fluorescence; Genetic; Grant; Green Fluorescent Proteins; Image; In Vitro; Life; Link; Location; Mammalian Cell; Methods; Molecular; Nuclear; Nucleotides; Physiological Processes; Protein Binding; RNA; RNA Binding; Randomized; Reporter; Reporting; Signal Transduction; Specificity; Structure; System; aptamer; biohazard detection; cellular imaging; detector; in vivo; novel; novel strategies; optical imaging; optical sensor; response; tool

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.

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。我们已经鉴定出可与绿色荧光蛋白结合并显着降低其荧光的 RNA 适体。鉴定出的适体结构以高亲和力与 GFP、eGFP、YFP 和 CFP 结合，但主要通过降低摩尔消光系数选择性地抑制 GFP 和 eGFP 的荧光发射 (KD = 14 nM)。在这里，我们建议利用荧光猝灭 RNA 适体作为灵活的、遗传可编码的分子检测系统。我们将优化核和细胞质 RNA 适体表达的遗传构建体（目标 1）。荧光蛋白结合结构将通过与第二个检测器结构域连接，用作二价 RNA 的变构调节报告组件（目标 2）；目标分子与 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 解除结合，并诱导强荧光信号。这样的系统可以为体外和体内细胞成像提供一种新方法，也可以适用于快速生物危害检测。细胞内和细胞间的生化信号传导是正常和异常功能的基础。光学成像和遗传规范的最新进展彻底改变了我们对复杂生理过程（例如发育和对疾病的反应）的理解。该提案旨在开发一种灵活且广泛适用的方法来构建基因编码光学传感器，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。