Fig. 5: Development of far-red NAD⁺ biosensors based on fluorescence intensity and fluorescence lifetime.

a, Schematic representation of ChemoD–NAD. b, Fluorescence intensity emission spectra of SiR-labeled ChemoD–NAD at different NAD⁺ concentrations. Means of three technical replicates are shown. c, NAD⁺ titration curves of ChemoD–NAD labeled with CPY, JF₆₃₅ or SiR. Data are shown as the means ± s.d. of the fluorescence intensity changes (ΔFI/FI₀; n = 3 technical replicates). ΔFI/FI₀ and C₅₀ values are summarized in Supplementary Table 9. d, Fluorescence lifetime decay curves of ChemoD–NAD_SiR in the presence of 1 mM NAD⁺ (+NAD⁺) or absence of NAD⁺ (–NAD⁺). e, Intensity-weighted average fluorescence lifetimes (τ) of ChemoD–NAD labeled with different fluorophores. Shown are the mean intensity-weighted average fluorescence lifetimes in the presence of 1 mM NAD⁺ (+NAD⁺) or absence of NAD⁺ (–NAD⁺) and the change in lifetime (Δτ; n = 3 technical replicates). f, NAD⁺ titration curves of ChemoD–NAD labeled with CPY, JF₆₃₅ or SiR. Shown are the means ± s.d. of the intensity-weighted average fluorescence lifetime changes (Δτ; n = 3 technical replicates). Δτ and C₅₀ values are summarized in Supplementary Table 10. g,h, Confocal images of U-2 OS cells expressing ChemoD–NAD labeled with CPY. Images are representative snapshots of the CPY fluorescence intensity channel (g) or average photon arrival time (APAT; h) before (–MNNG) and after (+MNNG) 100 µM MNNG treatment. Time course measurements of ChemoD–NAD_CPY fluorescence intensity normalized to 1 at t = 0 min (g; n = 86 cells from three biological replicates) and intensity-weighted average fluorescence lifetime (h; n = 55 cells from three biological replicates) in U-2 OS cells are shown next to the confocal images corresponding to the same treatments. Represented are the means (lines) and traces of single cells (dim lines). Addition of 100 µM MNNG is indicated with arrows; scale bars, 25 µm.

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