Figure 1. A highly responsive genetically encoded NAD⁺/AXP ratio sensor.

(A) Schematic representation of the NAD⁺ sensor FiNad. Fluorescent protein cpYFP was inserted into a monomer of NAD⁺(NADH)-binding bacterial protein T-Rex. Binding of NAD⁺ induces changes in protein conformation and fluorescence. (B) Fluorescence spectra of purified FiNad in the control condition (black), and saturated NAD⁺ (dark red). The excitation spectrum recorded at an emission wavelength of 535 nm has a maximum at 500 nm; the emission spectrum recorded at an excitation wavelength of 490 nm has a maximum at 518 nm. (C) Fluorescence responses of FiNad with excitation at 485 nm in the presence of different concentrations of NAD⁺ and its analogs (n=3). Data are normalized to the initial value. (D) FiNad sensor responds to the indicated [NAD⁺], but not to the NAD⁺/NADH ratio (n=3). Data are normalized to the fluorescence in the absence of NAD⁺. (E) FiNad fluorescence plotted against NAD⁺/AXP ratio at the indicated total adenine nucleotide concentration (n=3). Data are normalized to the initial value (1mM AXP). (F) The green/red ratio of mCherry-FiNad towards NAD⁺ or its precursors (n=3). Data are normalized to the initial value. For C, D and F, the total adenine nucleotide concentration was 1 mM. Data are the mean ± s.d. See also Figure S1, Table S1 and Table S2.