Orthogonal Bioluminescent Probes from Disubstituted Luciferins

Abstract

Bioluminescence imaging with luciferase-luciferin pairs is routinely used to monitor cellular functions. Multiple targets can be visualized in tandem using luciferases that process unique substrates, but only a handful of such orthogonal probes are known. Multiplexed studies require additional robust, light-emitting molecules. In this work, we report new luciferins for orthogonal imaging that comprise disubstituted cores. These probes were found to be bright emitters with various engineered luciferases. The unique patterns of light output also provided insight into enzyme-substrate interactions necessary for productive emission. Screening studies identified mutant luciferases that could preferentially process the disubstituted analogues, enabling orthogonal imaging with existing bioluminescent reporters. Further mutational analyses revealed the origins of substrate selectivity. Collectively, this work provides insights into luciferase-luciferin features relevant to bioluminescence and expands the number of probes for multicomponent tracking.

Figure 1.

Multicomponent bioluminescence imaging with engineered luciferases and luciferins. (A) Fluc catalyzes the oxidation of D-luc, producing photons of light. (B) Mutant luciferases preferentially process sterically modified analogs. Orthogonal pairs comprise enzymes that are bright with one analog, while dim with another. Substrate preference can be graphically depicted as shown in the cartoon plot. (C) Disubstituted luciferins examined in this work (right), alongside the relevant singly modified analogs (left). (D) Overlay of 4’,7’-MeLuc (docked as AMP ester conjugate, dark gray) in the active site of Fluc (PDB: 4G36). Residues within 5 Å of the methyl substituents (orange) are shown in blue.

Figure 2.

Disubstituted analogs produce light with Fluc. Analogs (0–1 mM) were incubated with (A) recombinant Fluc or (B) Fluc-expressing HEK293 cells. For (B), photon outputs were monitored over time and steady state emission values (t = 25 min) are shown. For (A)-(B), emission intensities are plotted as photon flux values on log scales. Error bars represent the standard error of the mean for n ≥ 3 experiments.

Figure 3.

Light emission of luciferin analogs incubated with a panel of mutant luciferases. (A) Mutant luciferase-expressing bacteria were lysed, plated in 96-well black plates, and incubated with 4′,7′-MeLuc (blue circles, 250 μM) or 4′-Br-7′-MeLuc (gray squares, 250 μM). Fluc-expressing bacteria were included in the screen, and the corresponding light emission values are highlighted in pink. The inset highlights a subset of mutants further analyzed in this work. (B) Residues of interest for orthogonal probe development. Analog 4′,7′-MeLuc is shown (as AMP ester conjugate, dark gray) bound in the Fluc active site (PDB: 4G36). The methyl groups are colored orange, while the sites targeted in mutants 53 and 54 are shown in blue. (C) Light emission from a subset of mutants and analogs. Saturating doses of 4′,7′-MeLuc (250 μM), 4′-Br-7′-MeLuc (250 μM), 4′-MeLuc (100 μM), or 4′-BrLuc (100 μM) were incubated with lysed bacterial cells expressing mutants 53 or 54. For each replicate in (A) and (C), a single lysate was split among the wells and treated with the various analogs. Emission intensities are shown as photon flux values on log scales. Error bars represent the standard error of the mean for n ≥ 3 experiments.

Figure 4.

Orthogonal substrate usage observed between mutants 53 and 54. Mutant 53 produces more light with disubstituted luciferins than singly modified analogs. Bacteria expressing mutant 53, mutant 54, or Fluc were lysed and incubated with 4’-Br-7’-MeLuc or a singly modified luciferin at saturating doses (100–250 μM). The light output for each enzyme-substrate reaction was measured. Comparative emission values are plotted as the ratio of disubstituted luciferin activity over the activity of each substrate shown, on a log scale. Error bars represent the standard error of the mean for n ≥ 3 experiments. For each replicate, a single lysate was split among three wells and treated with the various analogs.

Figure 5.

Screen to identify key residues involved in analog selectivity. (A) Library screening workflow. (B) Sample bioluminescence images from library screens with 4′-Br-7′-MeLuc, 4′-BrLuc, and D-luc. Selective colonies were selected for sequencing analysis. For each replicate, a single lysate was split among the wells and treated with the compounds shown.

Figure 6.

Substrate selectivity of frequently occurring mutants. (A) Frequency of residues observed in mutants that prefer 4’,7’-MeLuc over D-luc and 4’-MeLuc, and vice versa. (B) Frequency of residues observed in mutants that prefer 4’-Br-7’-MeLuc over D-luc and 4’-BrLuc, and vice versa. The pink heat maps represent mutants that preferred 4’,7’-MeLuc or 4’-Br-7’-MeLuc, while the blue heat map displays mutants that preferred either D-luc, 4’-BrLuc or 4’-MeLuc.

Figure 7.

Disubstituted luciferins are orthogonal to robust light-emitting luciferins, including (A) D-luc, (B) 4′-MeLuc, and (C) 7′-MeLuc. Mutants F247Y/S347G and 53 selectively processes the disubstituted analogs, while Fluc and mutant 87 prefer other luciferins. Bacterial cells expressing (A)-(B) mutant F247Y/S347G or Fluc or (C) mutant 53 or 87 were lysed and plated over a 96-well plate. Comparative emission values are plotted as the ratio of one luciferase over the other, on a log scale, for each substrate. Error bars represent the standard error of the mean for n ≥ 3 experiments. For each replicate, a single lysate was split among the wells and treated with each analog.

Scheme 1.

Synthesis of disubstituted luciferin analogs (A) 4′,7′-MeLuc and (B) 4′-Br-7′-MeLuc