Fig. 2.

Characterization of LUPIN. a Bioluminescence resonance energy transfer from Nluc to ruthenium is observed when SNAP-NLuc-DHFR was labeled with BG-(Ru)(PNA)-MTX (1). Emergence of a new band at 610 nm indicates BRET from Nluc to ruthenium (red line); in the presence of methotrexate, more of the sensor shifts into the open conformation, decreasing BRET efficiency (black line). b Fluorescence enhancement due to rhodamine uncaging by the LUPIN system is observed. In the presence of methotrexate, the reaction is partially inhibited due to lower BRET efficiency. In the absence of nucleic acid template, or in the absence of a covalent link between the synthetic linker and the protein construct, the reaction is marginal. Reaction conditions: SNAP-NLuc-DHFR labeled with BG-(Ru)(PNA)-MTX (1) (50 nM), PNA-PyRho (2) or PrPyRho (3) (0.5 µM), sodium ascorbate (10 mM), furimazine (100 µM), and methotrexate (100 µM). BG benzyl guanine. c Varying the concentration of the labeled SNAP-NLuc-DHFR protein (50, 10, and 2 nM) has a significant impact on luminescence decay and the rate of rhodamine release