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: 7465429
• 负责人: Michael I. Kotlikoff
• 金额: $ 18.87万
• 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/7465429
• 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）：该探索性赠款旨在开发一种新颖而有希望的细胞内成像系统。我们已经确定了与绿色荧光蛋白的荧光结合并显着减少的RNA适体。鉴定的适体结构与具有高亲和力的GFP，EGFP，YFP和CFP结合，但有选择地抑制GFP和EGFP（KD = 14 nm）的荧光发射，这在很大程度上通过摩尔灭绝系数的降低。在这里，我们建议将荧光淬灭RNA适体作为柔性，遗传编码的分子检测系统。我们将优化用于表达核和细胞质RNA适体的遗传构建体（AIM 1）。荧光蛋白结合结构将通过与第二个检测器结构域的链接（AIM 2）链接来用作二价RNA的变构调节报告基因组件；靶分子与RNA适体内检测器结构域的结合将导致从GFP与荧光淬灭RNA适体的脱落，并诱导强大的荧光信号。这样的系统可以提供一种新型的体外和体内细胞成像方法，也可以适应快速的生物危害检测。细胞内以及细胞之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已彻底改变了我们对复杂生理过程（例如发育和对疾病反应）的理解。该提案旨在开发一种灵活且广泛的方法，用于构建遗传编码的光学传感器，该方法将报告活细胞中特定分子的浓度，位置和结构，从而显着扩展了理解复杂细胞反应的工具。