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. ? ? ?
|Shui, Bo; Ozer, Abdullah; Zipfel, Warren et al. (2012) RNA aptamers that functionally interact with green fluorescent protein and its derivatives. Nucleic Acids Res 40:e39|
|Wang, Qi; Shui, Bo; Kotlikoff, Michael I et al. (2008) Structural basis for calcium sensing by GCaMP2. Structure 16:1817-27|