A higher spectral range of beetle bioluminescence with infraluciferin

Abstract

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH₂). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH₂). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH_2, including in human cells. iLH₂ was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH₂: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH₂, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH₂ λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase (D-LH₂ λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL-iLH₂ whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH_2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH₂ and engineer an improved infraluciferase (iluc) based on this mutant.

Keywords: bioluminescence; green; infraluciferin; luciferase; multicolor; near-infrared; spectral range.

FIGURE 1

Bioluminescence reactions of beetle luciferases. Bioluminescence with D-LH₂ (A) and D-iLH₂ (B) (4S)-2-[(E)-2-(6-hydroxy-1,3-benzothiazol-2-yl)ethenyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid; ATP: adenosine triphosphate. (C) Inset table of different subsets of mutations in thermostable mutants of Ppy Fluc: x2 Fluc (Willey et al., 2001), x5 Fluc (Law et al., 2006), and x11 Fluc (Jathoul et al., 2012a).

FIGURE 2

Specific activities of WT Ppy Fluc, thermostable Ppy-based Fluc mutants, Eluc, and CBR with DL-iLH₂. Specific activity of 0.16 μM enzymes at pH 7.3 in PEM buffer, 200 μM D-LH₂ or DL-iLH₂ and 2 mM ATP, and emission was captured for 3 min through the open filter in the PIO. p-values for t-test of activity with DL-iLH₂ between WT Fluc and x2 (>0.0001), x5 (0.0003), x11 (>0.0001), Eluc (0.0001), and CBR (0.0002).

FIGURE 3

Expanded bioluminescence spectral range with infraluciferin. (A) Bioluminescence spectra of different luciferases with D-LH₂ and DL-iLH₂: 5 μM enzymes were assayed with 200 μM luciferins and 2 mM ATP, and light was captured using the Clariostar instrument. For ease of visualization, x5 and x11 Fluc spectra with D-LH₂ are omitted but are near identical to WT Fluc (Reference, Table 1). (B) Illustration of spectral separation between Eluc and CBR with DL-iLH₂ measured in the PIO with 0.5 μM enzymes (details as in Supplementary Figure S5).

FIGURE 4

Effect of Fluc mutations E354R and D357Y on activity and color with DL-iLH₂. (A) Quantitative bioluminescence spectra showing that E354R leads to significant improvement in activity with DL-iLH₂. (B) Normalized bioluminescence spectra of WT Ppy Fluc, E354R, and D357Y with D-LH₂ and DL-iLH₂.

FIGURE 5

Activity of purified Flucs and mutants with both substrates. Specific activity and emission peak wavelengths of selected R354 and/or D357 mutants of WT, x5, and x11 Flucs. 150μM D-LH₂ or 15 μM DL-iLH₂ and 2 mM ATP were used to saturate 0.167 and 0.0167 μM Flucs, respectively, and light emission was captured using the PIO. The assay was then repeated with the blood phantom.

FIGURE 6

Bioluminescence yields and spectra from transfected HEK 293 cells. (A) EGFP normalized light yields from whole HEK cells. 1mM D-LH₂ K⁺ salt or DL-iLH₂ Me ester was added to initiate luminescence, and emission was captured over 5 min at 30°C in the PIO (Biospace Labs, Paris, France). Emission with both substrates was observed to take approximately 1 min to reach a plateau, and light yields given were from 1 min after addition of substrates. Background signals were obtained from triplicate ROIs placed in areas of images containing no wells. (B) Normalized bioluminescence spectra obtained from whole transfected HEK cells expressing different variants with (B) D-LH₂ and (C) DL-iLH₂ Me ester imaged in the PIO.

FIGURE 7

Imaging and unmixing HEK 293 cells expressing dual mixes of ilucG3 and ilucR. Substrates were dispensed with a multiwell pipette and, after 5 min, were imaged in successive filters on the PIO (622, 722, and 797 nm band-passes (BP)). Regions of interest were analyzed for photon flux at different wavelengths using the M3 Vision software. (A) Quantitative bioluminescence spectra from variants. (B) Increasing percentage of ilucG3 imaged through 622, 722 and 797BP filters.