Fig. 1. Principles of (a) traditional FRET, BRET and (b) BASFI in the context of protein-protein interaction.((a) An external light source or bioluminescence reaction creates the donor in FRET or BRET, respectively, and the immediate ET sensitized emission (ET-SE) is collected to measure ET efficiency. This results in severe spectral bleedthrough (ABT and DBT) in FRET and feeble ET-SE signal in BRET. (b) In BASFI, firstly ET drives acceptor ground state photoswitching from dark (left) to bright state (middle). Then the bright state population is directly imaged by an external light (right). Note the bright LEE emission and the absence of bleedthroughs in BASFI. DBT: Donor BleedThrough; ABT: Acceptor BleedThrough; ET-SE: Energy-Transfer-Sensitized Emission; CTZ: coelenterazine and its derivatives; LEE: Laser Elicited Emission.
Research-Bioluminescence Assisted Switching and Fluorescence Imaging.
Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are two major biophysical techniques for studying nanometer scale dynamics such as protein-protein interaction within living cells. Spectroscopically, both techniques rely on RET coupled concurrent acceptor photoemission. Consequently, FRET provides high photon flux but suffers from spectral bleedthroughs, whereas BRET is nearly bleedthrough-free but its feeble emission signal hinders high-resolution cell imaging (Fig. 1a). To circumvent this difficulty, we have explored a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI): the donor is the bioluminescent Renilla luciferase RLuc8 and its proper substrates, and the acceptor is the photochromic protein Dronpa.
When in close proximity, RET from RLuc8 during catalysis switches Dronpa to its bright state, whose fluorescence can be imaged subsequently with an external laser (Fig. 21 b). Such temporal decoupling between the RET process and the imaging process, made possible by the introduced photochromism of the acceptor, not only bypasses the spectral bleedthroughs of FRET but also drastically elevates the photon flux of BRET to achieve high-resolution protein-protein interaction images. We first demonstrated the principle of BASFI with Dronpa-RLuc8 fusion constructs, and then applied it to image drug-inducible intermolecular FKBP-FRB interactions in live cells (shown in Fig. 22) with high sensitivity, resolution and specificity . Integrating the advantages of FRET and BRET, BASFI will be a valuable tool for various biophysical studies.
Fig. 2.Live cell intermolecular BASFI of Rap induced L-FKBP2-RLuc8 and DG1-FRB association in HEK293T cells.(a)-(d): Ratio images of BRET2-switched-on population of DG1 after incubating with different substrates for 10 minutes. (a) me-eCTZ; (b) me-eCTZf; (c) me-CTZ; (d) CTZ400a. (e) Control images without addition of substrates. (f) Control images using the substrate me-eCTZ but without Rap.
1. L. Zhang, F. Xu, Z. Chen, X. Zhu and W. Min. "Bioluminescence assisted switching and fluorescence imaging (BASFI)", J. Phys. Chem. Lett. 4, 3897 (2013).[PDF]