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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.

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Figure 1: Imaging and monitoring the NAD+/NADH redox state in single living cells.
Figure 2: SoNar-based high-throughput screening for compounds affecting cell metabolism.
Figure 3: Imaging and monitoring the NAD+/NADH redox state in living mice.

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Acknowledgements

We thank G. Yellen (Harvard Medical School, Boston, MA) for the Peredox vector; M. Chen, S. Lan, S. Han, Y. Ci, R. Yang, L. Yue and Y. Wang for technical assistance; and J. Zou for editing the manuscript. This research was supported by the 973 Program (2013CB531200 to Y.Y.), NSFC (91313301, 31225008, 31071260, 31170815, 31470833 and 91013012 to Y.Y.), the Specialized Research Fund for the Doctoral Program of Higher Education (20100074110010 to Y.Y.), the Shanghai Science and Technology Commission (12JC1402900 and 11DZ2260600 to Y.Y., and 15YF1402600 to Y. Zhao), the Dawn Program of the Shanghai Education Commission (11SG31 to Y.Y.), the Lift Engineering for Young Talent of China Association for Science and Technology (to Y. Zhao), the State Key Laboratory of Bioreactor Engineering (to Y.Y.), the 111 Project (B07023 to Y.Y.), the Fundamental Research Funds for the Central Universities (to Y.Y. and Y. Zhao) and the National Institutes of Health (grants HL061795, HL048743, HL108630 and GM107618 to J.L.).

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Authors

Contributions

Y.Y. and Y. Zhao conceived and designed the in vitro and in vivo imaging and detection, and metabolic screening experiments. Y. Zhao, A.W., Y. Zou and N.S. performed experiments. Y.Y., Y. Zhao and J.L. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Yi Yang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Properties of the Genetically Encoded NADH sensor Frex and NAD+/NADH Sensor SoNar.

(a) The response of Frex sensor to various NADH concentrations at the indicated [NAD+].

(b) Responses of SoNar and Peredox to different NAD+/NADH ratios (Figure from our previous work1). For SoNar, the total nicotinamide adenine dinucleotide concentration was 400 μM. Fluorescence ratios were normalized to the value in the presence of NAD+ (400 μM), at pH 7.4. For Peredox, fluorescence ratios were normalized to the control condition in the absence of pyridine nucleotides at pH 7.2, with 230 μM NAD+ (data redrawn from Yellen's work2).

(c) Fluorescence intensities of SoNar and cpYFP with excitation at 420 nm or 485 nm, and emission at 528 nm. Data normalized to the fluorescence at pH 7.4 (Figure from our previous work1).

(d) Fluorescence intensity of SoNar when excited at 420 nm plotted against the NAD+/NADH ratio at the indicated pH (Adapted from our previous work1).

(e and f) Un-normalized (e) and normalized (f) ratio of SoNar fluorescence excited at 420 nm and 485 nm at the indicated pH plotted against the NAD+/NADH ratio. For f, the data was normalized to the scale of 0-1 to demonstrate that SoNar dynamic range and KNAD+/NADH are more pH resistant (For e, Adapted from our previous work1; For f, Figure from our previous work1).

Supplementary Figure 2 Compounds used in high-throughput screening.

(a) 23 commercial compound libraries

(b) Relative proportion of compounds that markedly affected the intracellular NAD+/NADH ratio across different chemical libraries (Figure from our previous work1).

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Zhao, Y., Wang, A., Zou, Y. et al. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state. Nat Protoc 11, 1345–1359 (2016). https://doi.org/10.1038/nprot.2016.074

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