title: In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD(+)/NADH redox state.
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.
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.|Optogenetics &Molecular Imaging Interdisciplinary Research Center, CAS Center for Excellence in Brain Science, East China University of Science and Technology, Shanghai, China.|Collaborative Innovation Center of Genetics and Development, East China University of Science and Technology, Shanghai, China.|Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Yuzheng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.|Optogenetics &Molecular Imaging Interdisciplinary Research Center, CAS Center for Excellence in Brain Science, East China University of Science and Technology, Shanghai, China.|Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Aoxue
authors.initials: A
authors.lastname: Wang
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.|Optogenetics &Molecular Imaging Interdisciplinary Research Center, CAS Center for Excellence in Brain Science, East China University of Science and Technology, Shanghai, China.|Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Yejun
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.|Optogenetics &Molecular Imaging Interdisciplinary Research Center, CAS Center for Excellence in Brain Science, East China University of Science and Technology, Shanghai, China.|Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Ni
authors.initials: N
authors.lastname: Su
authors.affiliation: Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Loscalzo
authors.affiliation: Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.|Optogenetics &Molecular Imaging Interdisciplinary Research Center, CAS Center for Excellence in Brain Science, East China University of Science and Technology, Shanghai, China.|Collaborative Innovation Center of Genetics and Development, East China University of Science and Technology, Shanghai, China.|Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
authors.forename: Yi
authors.identifier: 0000-0001-7896-1184
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004734:Energy Metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009243:NAD / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods*; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD