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.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism

“…Genetically encoded fluorescent sensors capable of live cell monitoring were developed for pyridine nucleotides such as NADH 9–12 , NAD +13 , and NADP +14 . Among them, SoNar is a highly responsive sensor for the NADH/NAD + ratio with superior properties useful for live cell and in vivo studies 10, 15, 16 . We designed the iNap NADPH sensors by engineering SoNar’s binding site to switch ligand selectivity from NADH to NADPH.…”

The redox requirements of proliferating mammalian cells

“…NADPH is used in reduction reactions, and cells maintain a high NADPH:NADP + ratio to ensure that these reactions are favorable (7,8). Conversely, NAD + is used as an electron acceptor to oxidize biosynthetic intermediates, and the NADH:NAD + ratio is typically very low in proliferating cells (9,10). The exact values of these ratios differ between cellular compartments and across growth conditions and impact which enzyme-catalyzed reactions are favorable in different contexts.…”

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD+ with Genetically Encoded Fluorescent Biosensors

“…The responses of the two other genetically encoded NADH/NAD + biosensors, Frex 15 and SoNar 16, 33 , were also studied by time-resolved fluorescence (see Figs 3a and 4a). In contrast to the GFP-based Peredox, Frex and SoNar are YFP-based.…”

“…The protocols and preparation details can be found in the references (Peredox 14 , Frex 15, 35 , and SoNar 16, 33 ). Peredox was diluted in MOPS buffer (100 mM MOPS, 50 mM KCl, 5 mM NaCl) to 0.1 μM at pH 7.0.…”