Theme：In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state

Host：Dr. Yu-Zheng Zhao

Time：23th June, 2020, 10:00-11:40

Location: Room 418, Med-X Research Institute, Xuhui Campus, SJTU

Abstract: 

NADH and its oxidized form NAD(+) have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD(+)/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as SoNar (sensor of NAD(H) redox). The chimeric SoNar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein from Thermus aquaticus (T-Rex). It functions by binding to either NAD(+) or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of SoNar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing SoNar in mice. Typically, the approximate time frame for fluorescence imaging of SoNar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism.

Biography: 

Dr. Yu-Zheng Zhao's main research areas are the development of in situ detection and imaging techniques for cell metabolism and the development of drug candidates for metabolic diseases. In recent years, the first generation of cell metabolism fluorescent probe Frex and the second generation of cell metabolism fluorescent probe SoNar have been developed successively for the core intracellular metabolite NADH, which has achieved in vivo, living cells and various subcellular structures. The real-time dynamic and specific quantitative detection and imaging of NADH metabolism in China has solved a key technical bottleneck in cell metabolism research. Relevant research results have an important impact on the international level. His developed NADH detection technology has used in more than 100 domestic and international institutions such as Harvard University, Massachusetts Institute of Technology, Oxford University, Cambridge University, Chinese Academy of Sciences and Peking University.