Fig. 4: High substrate specificity of de novo luciferases allows multiplexed bioassay.
a, Chemical structures of coelenterazine substrate analogues. b, Normalized activity of LuxSit-i on selected luciferin substrates. Luminescence image (top) and signal quantification (bottom) of the indicated substrate in the presence of 100 nM LuxSit-i. LuxSit-i has high specificity for the design target substrate, DTZ. c, Heat map visualization of the substrate specificity of LuxSit-i; Renilla luciferase (RLuc); Gaussia luciferase (GLuc); engineered NLuc from Oplophorus luciferase; and the de novo luciferase (HTZ3-G4) designed for h-CTZ. The heat map shows the luminescence for each enzyme on each substrate; values are normalized on a per-enzyme basis to the highest signal for that enzyme over all substrates. d, The luminescence emission spectrum of LuxSit-i-DTZ (green) and RLuc-PP-CTZ (purple) can be spectrally resolved by 528/20 and 390/35 filters (shown in dashed bars) and only recognize the cognate substrate. e, Schematic of the multiplex luciferase assay. HEK293T cells transiently transfected with CRE-RLuc, NF-κB-LuxSit-i and CMV-CyOFP plasmids were treated with either forskolin (FSK) or human tumour necrosis factor (TNF) to induce the expression of labelled luciferases. f,g, Luminescence signals from cells can be measured under either substrate-resolved or spectrally resolved methods by a plate reader. f, For the substrate-resolved method, luminescence intensity was recorded without a filter after adding either PP-CTZ or DTZ. g, For the spectrally resolved method, both PP-CTZ and DTZ were added, and the signals were acquired using 528/20 and 390/35 filters simultaneously. In f and g, the bottom panel indicates the addition of FSK or TNF. Luminescence signals were acquired from the lysate of 15,000 cells in CelLytic M reagent, and the CyOFP fluorescence signal was used to normalize cell numbers and transfection efficiencies. All data were normalized to the corresponding non-stimulated control. Data are mean ± s.d. (n = 3).
