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Improved HaloTag Ligand Enables BRET Imaging With NanoLuc


Improved HaloTag Ligand Enables BRET Imaging With NanoLuc

Ovia Margaret Thirukkumaran et al. Front Chem.


Bioluminescence resonance energy transfer (BRET) from an exceptionally bright luciferase, NanoLuc, to a fluorescent HaloTag ligand is gaining momentum to monitor molecular interactions. The recommended use of HaloTag618 ligand for the NanoLuc-HaloTag BRET pair is versatile for ensemble experiments due to their well-separated emission bands. However, this system is not applicable for single-cell BRET imaging because of its low BRET efficiency and in turn weak acceptor signals. Here we explored the unprecedented potential of rhodamine based HaloTag ligands, containing azetidine rings, as BRET acceptors. Through a comprehensive evaluation of various commercial and Janelia Fluor HaloTag ligands for improved BRET efficiency and minimal donor signal bleed-through, we identified JF525 to be the best acceptor for microscopic BRET imaging. We successfully employed BRET imaging with JF525 to monitor the interaction of protein kinase A catalytic and regulatory subunit. Single-cell BRET imaging with HaloTag JF525 can henceforth open doors to comprehend and interpret molecular interactions.

Keywords: BRET imaging; HaloTag; Janelia Fluor dyes; NanoLuc; PKA.


Figure 1
Figure 1
Spectra of NanoLuc and HaloTag ligands. (A) Normalized absorption spectra of HaloTag ligands. The spectra of the different ligands are in dotted lines (OG, bluish green, JF503, yellowish green, JM525, yellow, JF549, violet, TMR, purple, Halo618, red) and overlaid with the normalized luminescence spectrum of NanoLuc (cyan solid line). The filled region indicates the overlap area (the products of two spectra). (B) Luminescence spectra of Halo-NanoLuc conjugated with Halo-ligands. Spectra were normalized with the donor peak at 460 nm. Dotted blue lines indicate the acceptor spectrum calculated by subtracting the spectrum for only NanoLuc (cyan) from that with the ligand. The wavelength where the acceptor spectrum intersects the NanoLuc spectrum (SD:A) is denoted with a black line. Rbt is the ratio of yellow area over sum of yellow and cyan areas, whereas FD is the ratio of yellow area over sum of the yellow area and the area colored with the color code of respective ligands.
Figure 2
Figure 2
BRET imaging in living cells. (A) Images of CHO-K1 cells expressing Halo-NanoLuc. Images were acquired after loading of respective ligands (OG, JF503, JF525, JM549, TMR, and Halo618) or in the absence of ligands (blank), using a dichroic mirror indicated to separate donor (upper) and acceptor (lower) windows. All images were obtained with identical acquisition settings (APON 60XOTIRF, NA = 1.49, exposure time = 0.75 s). Scale bar = 10 μm. (B,C) Linear regression analyses of the acceptor vs. donor signal intensities for green-shifted (B) or orange-red ligands (C). The color codes of ligand-loaded cells are same as in Figure 1. Cells in the absence of ligands (blank) are shown in black. (D) Mean RA/D ± SEM calculated from the cells labeled with different ligands. n ≥ 70.
Figure 3
Figure 3
Dissociation of PKA subunits observed by BRET. (A) Interaction of NL-RS and CS-HT modeled with the crystal structures (PDB files: 2QBVS, 2F7E, 5IBO, 5Y2Y). The regulatory subunit is shown in purple, catalytic subunit in wheat, NanoLuc in cyan, and HaloTag in greenish-yellow. (B) Schematic explaining the decrease in BRET upon dissociation of PKA subunits. The tetrameric structure of tagged NL-RS and CS-HT under resting condition (left) is labeled with HaloTag ligands (middle) and stimulated with forskolin to dissociate the interaction of RS and CS (right). (C) Luminescence images of NIH3T3 cells co-expressing NL-RS and CS-HT. Images were acquired after loading respective HaloTag ligands (JF525 and Halo618). All images were acquired with identical camera settings. Scale bar = 10 μm. (D) Representative intensity traces of donor and acceptor signals and (E) Mean SNR ± SEM for cells labeled with either JF525 or Halo618. (F) Time traces of acceptor/donor emission ratios showing dissociation of PKA subunits NL-RS and CS-HT upon forskolin stimulation (indicated by black arrow). (G) Box plot showing ΔRA/D values for cells labeled with respective HaloTag ligands. n = 8.

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