Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides

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Abstract

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.

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All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, & 4, and Appendix 1-Figures 1, 3, & 7.

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Olivier Sallin

Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Competing interests
Olivier Sallin, has filed a patent application (WO2016131833A1) on the design and use of sensors for the detection of NAD(P).
Luc Reymond

Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Competing interests
Luc Reymond, has filed a patent application (WO2016131833A1) on the design and use of sensors for the detection of NAD(P).
Corentin Gondrand

Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany

Competing interests
No competing interests declared.
Fabio Raith

Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany

Competing interests
No competing interests declared.
Birgit Koch

Department of Chemical Biology, Max-Planck-Institute of Medical Research, Heidelberg, Germany

Competing interests
No competing interests declared.
Kai Johnsson

Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

For correspondence
kai.johnsson@epfl.ch

Competing interests
Kai Johnsson, has filed a patent application (WO2016131833A1) on the design and use of sensors for the detection of NAD(P).

"This ORCID iD identifies the author of this article:" 0000-0002-8002-1981

Funding

Max-Planck-Gesellschaft (Institutional support and open-access funding)

Kai Johnsson

Ecole Polytechnique Federale de Lausanne (Institutional support and open-access funding)

Corentin Gondrand

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

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.