Near-Infrared Imaging of Steroid Hormone Activities Using Bright BRET Templates

Abstract

Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the NIR-BRET templates through the characterization of many coelenterazine (CTZ)-marine luciferase combinations. As a result, we found that NLuc-DBlueC and ALuc47-nCTZ combinations showed luminescence in the blue emission wavelength with excellent BL intensity and stability, for example, the NLuc-DBlueC and ALuc47-nCTZ combinations were 17-fold and 22-fold brighter than their second highest combinations, respectively, and were stably bright in living mammalian cells for at least 10 min. To harness the excellent BL properties to the NIR-BRET systems, NLuc and ALuc47 were genetically fused to fluorescent proteins (FPs), allowing large "blue-to-red" shifts, such as LSSmChe, LSSmKate2, and LSSmNep (where LSS means Large Stokes Shift). The excellent LSSmNep-NLuc combination showed approximately 170 nm large resonance energy shift from blue to red. The established templates were further utilized in the development of new NIR-BRET systems for imaging steroid hormone activities by sandwiching the ligand-binding domain of a nuclear receptor (NR-LBD) between the luciferase and the FP of the template. The NIR-BRET systems showed a specific luminescence signal upon exposure to steroid hormones, such as androgen, estrogen, and cortisol. The present NIR-BRET templates are important additions for utilizing their advantageous imaging of various molecular events with high efficiency and brightness in physiological samples.

Keywords: bioluminescence; bioluminescence resonance energy transfer (BRET); blue-to-red shift; coelenterazine; fluorescent protein; imaging; luciferase; near infrared; steroid.

Figure 1

(A) Chemical structures of conventional or newly synthesized CTZ derivatives: nCTZ, native coelenterazine; DBlueC, DeepBlueC; Ome, 6-ome-CTZ. They commonly share an imidazopyrazinone backbone that is highlighted in blue. (B) Relative and normalized BL spectra of selected CTZ derivatives with marine luciferases, ALuc16, ALuc47, and NanoLuc (NLuc). The representative full width at half maximum (FWHM) values were annotated with cyan color.

Figure 2

(A) The time course of the BL intensities of selected marine luciferases reacted with conventional and newly synthesized CTZ derivatives. (B) The BL images of live COS-7 cells stably expressing click beetle luciferase red (CBLuc red), NLuc, or ALuc47 on a 6-channel microslide in the presence of DBlueC or CTZ (upper panel). The corresponding fold intensities of the COS-7 cells highlighting their substrate specificity (lower panel). The p-values (student’s t-test) are ** < 0.01 and * < 0.1, respectively.

Figure 3

(A) Schematic diagram of cDNA constructs encoding various combinations of fluorescent protein (FP) and luciferase, designed for BRET imaging. Inset ‘a’ shows the typical working mechanism of the BRET probes after expression. (B) The BL images of various BRET probes in the presence of the specific substrate, CTZ for ALuc47-bearing probes (left side), or DBlueC for NLuc-bearing probes (right side). The images were captured with open, 640-nm, or 700 nm band-pass filter (n = 4). The asterisks (*) highlight brighter probes. (C) The BL spectra of LSSmNep_NLuc in various buffer conditions. The red shadow highlights the red and NIR region of the spectra.

Figure 4

cDNA constructs encoding various BRET probes for imaging steroid hormones. (A) A schematic diagram of BRET probes sandwiching AR HLBD. (B) A schematic diagram of BRET probes sandwiching GR HLBD. (C) A schematic diagram of BRET probes sandwiching ER HLBD. (D) The chemical structures of steroid hormones and their antagonists.

Figure 5

Initial screening of potential BRET probes for imaging steroid hormones. The upper panel shows the optical responses of each BRET probe in live COS-7 cells, and the lower panel shows the same in cell lysates. (A) Variance in the optical intensities of BRET probes sandwiching AR HLBD, stimulated by vehicle or DHT. (B) Variance in the optical intensities of BRET probes sandwiching GR HLBD, which were stimulated by vehicle or cortisol. (C) Variance in the optical intensities of BRET probes sandwiching ER HLBD, which were stimulated by vehicle, E2, or OHT. Abbreviations: AR HLBD, The hinge and ligand binding domain of human androgen recepter; ER HLBD, The hinge and ligand binding domain of human estrogen recepter; GR HLBD, The hinge and ligand binding domain of human glucocorticoid recepter; 0-9GS, Glycine and serine linker in the size of 1–9; LXX, An LXXLL motif (where L is leucine and X is any amino acid); veh, vehicle; DHT, 5α-dihydrotestosterone; E2, 17β-estradiol; and OHT, 4-Hydroxytamoxifen. The asterisks (*) highlight the best S/B ratio probes in the groups.

Figure 6

Ligand specificity of selected NIR BL imaging probes stably expressed in mammalian cells. (A) NIR BL intensities of the selected imaging probes in live cells after stimulation using various steroid hormones. (B) NIR BL intensities of the selected imaging probes in the lysates after stimulation using various steroid hormones. (C) Dose–response curves of LSSmNep-SH2-ER-NLuc (left) and LSSmNep-AR-NLuc (right) after stimulation of varying concentrations of steroid hormones. Inset ‘a’ illustrates the working mechanism of LSSmNep-SH2-ER-NLuc. Ligand-activated ER LBD is phosphorylated and recognized by the adjacent SH2 domain. The fold-up structure exerts enhanced NIR BL intensities. The p-values (student’s t-test) are ** < 0.01 and * < 0.1, respectively.