Genetically encoded fluorescent indicator for imaging NAD+/NADH ratio changes in different cellular compartments

Dmitry S. Bilan, Mikhail E. Matlashov, Andrey Yu Gorokhovatsky, Carsten Schultz, Grigori Enikolopov, Vsevolod V. Belousov

Research output: Contribution to journalArticlepeer-review

93 Scopus citations


Background The ratio of NAD+/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD+/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD+/NADH are fundamentally new approach for studying the NAD+/NADH dynamics. Methods We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy. Results The sensor, named RexYFP, reports changes in the NAD+/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD+/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD+/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore. Conclusion RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments. General significance RexYFP has several advantages over existing NAD+/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.

Original languageEnglish (US)
Pages (from-to)951-957
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Issue number3
StatePublished - Mar 2014
Externally publishedYes


  • Fluorescent probe
  • NAD/NADH ratio
  • Redox sensor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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