Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides

Elife. 2018 May 29;7:e32638. doi: 10.7554/eLife.32638.


We introduce a new class of semisynthetic fluorescent biosensors for the quantification of free nicotinamide adenine dinucleotide (NAD+) and ratios of reduced to oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) in live cells. Sensing is based on controlling the spatial proximity of two synthetic fluorophores by binding of NAD(P) to the protein component of the sensor. The sensors possess a large dynamic range, can be excited at long wavelengths, are pH-insensitive, have tunable response range and can be localized in different organelles. Ratios of free NADPH/NADP+ are found to be higher in mitochondria compared to those found in the nucleus and the cytosol. By recording free NADPH/NADP+ ratios in response to changes in environmental conditions, we observe how cells can react to such changes by adapting metabolic fluxes. Finally, we demonstrate how a comparison of the effect of drugs on cellular NAD(P) levels can be used to probe mechanisms of action.

Keywords: Nicotinamide adenine dinucleotides; biochemistry; biosensor; chemical biology; human; live-cell imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques*
  • Cell Line, Tumor
  • Cell Nucleus / metabolism
  • Cytosol / metabolism
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Mice
  • Mitochondria / metabolism*
  • NAD / analysis
  • NAD / metabolism*
  • NADP / analysis
  • NADP / metabolism*
  • NIH 3T3 Cells
  • Osteoblasts / metabolism
  • Osteoblasts / ultrastructure
  • Oxidation-Reduction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Rhodamines / chemistry
  • Rhodamines / metabolism
  • Sulfamethoxazole / metabolism
  • Sulfapyridine / metabolism


  • Fluorescent Dyes
  • Recombinant Fusion Proteins
  • Rhodamines
  • NAD
  • NADP
  • tetramethylrhodamine
  • Sulfamethoxazole
  • Sulfapyridine

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.