**4. Bioluminescence resonance energy transfer**

Bioluminescence Resonance Energy Transfer (BRET) technology involves the nonradioactive transfer of energy between donor and acceptor molecules by the Förster mechanism (46). The energy transfer primarily depends on the following: (1) an overlap between the emission and excitation spectra of the donor (bioluminescence) and acceptor (Fluorophore or a fluorescent protein) molecules, respectively; (2) the proximity of < 100 Å between the donor and the acceptor entities; and (3) the conformational orientation light emission with the acceptance end of the fluorescence entity. As BRET-based technology assumes more prominent roles in the field of studying PPIs, many commercial vendors are developing new instrumentations for measuring BRET ratios, which are generally low-intensity signals. BRET measurements are usually obtained with a microplate reader equipped with specific filter sets for detection of the donor and acceptor emission peaks. This cellular assay has been applied to real-time imaging of cells, high-throughput screening of drugs, and small animal and plant models. There are several combination of BRET involving Renilla luciferase and green fluorescent protein and Firefly luciferase with variants of red fluorescent proteins developed for studying proteinprotein interactions. The BRET2 system (Biosignal Packard Montreal, Canada) using renilla luciferase (RLUC) as a bioluminescent donor and mutant GFP2 as a fluorescent acceptor was

Bioluminescent Proteins: High Sensitive Optical Reporters

cells. *Anal Chem Vol.76*, No.8:pp.2181-2186.

cells. *Mol Endocrinol Vol.17*, No.4:pp.589-599.

*Embo J Vol.9*, No.13:pp.4207-4213.

*Methods Vol.19*, No.2:pp.278-305.

mice. *J Biomed Opt Vol.9*, No.3:pp.578-586.

research. *Ilar J Vol.42*, No.3:pp.219-232.

No.8:pp.2702-2709.

*U S A Vol.82*, No.23:pp.7870-7873.

living mammals. *Nat Med Vol.4*, No.2:pp.245-247.

interactions. *Nucleic Acids Res Vol.2*, No.10:pp.1951-1965.

No.5:pp.499-518.

for Imaging Protein-Protein Interactions and Protein Foldings in Living Animals 75

Artemov D, Mori N, Ravi R, and Bhujwalla ZM. (2003). Magnetic resonance molecular imaging of the HER-2/neu receptor. *Cancer Res Vol.63*, No.11:pp.2723-2727. Awais M, Sato M, Sasaki K, and Umezawa Y. (2004). A genetically encoded fluorescent

Bai Y, and Giguere V. (2003). Isoform-selective interactions between estrogen receptors and

Beck V, Rohr U, and Jungbauer A. (2005). Phytoestrogens derived from red clover: an

Becker T, Weber K, and Johnsson N. (1990). Protein-protein recognition via short

Beeckmans S. (1999). Chromatographic methods to study protein-protein interactions.

Bhaumik S, and Gambhir SS. (2002a). Optical imaging of Renilla luciferase reporter gene expression in living mice. *Proc Natl Acad Sci U S A Vol.99*, No.1:pp.377-382. Bhaumik S, and Gambhir SS. (2002b). Optical imaging of Renilla luciferase reporter gene expression in living mice. *Proc Natl Acad Sci USA Vol.99*, No.1:pp.377-382. Bhaumik S, Lewis XZ, and Gambhir SS. (2004). Optical imaging of Renilla luciferase,

Bode J, and Willmitzer. (1975). Application of fluorescamine to the study of protein-DNA

Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene

antagonism in the oestrogen receptor. *Nature Vol.389*, No.6652:pp.753-758. Burt BM, Humm JL, Kooby DA, Squire OD, Mastorides S, Larson SM, and Fong Y. (2001).

Cherry SR, and Gambhir SS. (2001). Use of positron emission tomography in animal

Contag CH, Jenkins D, Contag PR, and Negrin RS. (2000). Use of reporter genes for optical measurements of neoplastic disease in vivo. *Neoplasia Vol.2*, No.1-2:pp.41-52. Contag CH, and Ross BD. (2002). It's not just about anatomy: in vivo bioluminescence

Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P, Stevenson DK, and

De A, Ray P, Loening AM, and Gambhir SS. (2009). BRET3: a red-shifted bioluminescence

de Wet JR, Wood KV, Helinski DR, and DeLuca M. (1985). Cloning of firefly luciferase

bioluminescent reporter. *Photochem Photobiol Vol.66*, No.4:pp.523-531. Contag PR, Olomu IN, Stevenson DK, and Contag CH. (1998). Bioluminescent indicators in

experimental colorectal cancer. *Neoplasia Vol.3*, No.3:pp.189-195.

indicator capable of discriminating estrogen agonists from antagonists in living

steroid receptor coactivators promoted by estradiol and ErbB-2 signaling in living

alternative to estrogen replacement therapy? *J Steroid Biochem Mol Biol Vol.94*,

amphiphilic helices; a mutational analysis of the binding site of annexin II for p11.

synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living

GL, Gustafsson JA, and Carlquist M. (1997). Molecular basis of agonism and

Using positron emission tomography with [(18)F]FDG to predict tumor behavior in

imaging as an eyepiece into biology. *J Magn Reson Imaging Vol.16*, No.4:pp.378-387.

Benaron DA. (1997). Visualizing gene expression in living mammals using a

resonance energy transfer (BRET)-based integrated platform for imaging proteinprotein interactions from single live cells and living animals. *FASEB J Vol.23*,

cDNA and the expression of active luciferase in Escherichia coli. *Proc Natl Acad Sci* 

adapted for expression in mammalian cells and characterized by a significantly red-shifted Stokes shift that emits transferred energy at 508 nm. The resonance energy transfer from the reaction of the reconstructed RLUC protein with its substrate Deep Blue Coelenterazine (DBC) excites the GFP2 protein, as the two fused proteins Id and MyoD, or FKBP12 and FRB which interact in the presence of a small molecule mediator (rapamycin). Our lab also demonstrated the ability to detect signal from PPIs in cultured cells, as well as from the surface and deeper tissues of small living animals with implanted cells over expressing the fusion constructs (For further details, read (De et al., 2009; Dragulescu-Andrasi et al., 2011).

Our lab has recently showed that the BRET2 assay sensitivity can be significantly improved by using RLUC mutants with improved quantum efficiency and/or stability (eg, RLUC8 and RLUCM) as a donor. To extend the time of light measurement, we also developed CLZ400 (also known as bisdeoxycoelenterazine) analogs, showing that signal from our improved BRET2 vector can be monitored for up to 6 hours. This approach, currently undergoing continuing validation, should have important implications for the study of PPIs in cells maintained in their natural environment, particularly if it can be effectively applied for the evaluation of new pharmaceuticals. Most recently, further advances in this field have led us to develop a high photon efficiency, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant RLUC (RLUC8). This new BRET fusion protein (BRET3) exhibits a several fold improvement in light intensity in comparison to existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564 nm peak wavelength) of any reported bioluminescence protein that uses its natural coelenterazine substrate, a benefit that can be demonstrated at various tissue depths in small animals.

#### **5. Future directions in bioluminescence imaging**

Molecular imaging has been recognized as an important and exciting area of bio-medical research, mainly because of its ability to visually represent, characterize, and quantify biological processes in living subjects. Techniques such as Positron Emission Tomography (PET), Single-photon Emission Computed Tomography (SPECT), and Magnetic Resonance Imaging (MRI) have been extensively used in the clinic for several diagnostic and disease monitoring processes; all these systems explore intracellular proteins or other molecules as probes for the signal. Reporter genes (Bioluminescence, fluorescence, and PET), on the other hand, are capable of precisely monitoring sub-cellular processes and their native functional actions in cells, and imaging them in living animals. Challenges, however, remain in delivering these proteins in cells without perturbing the cellular microenvironments. Another obstacle is in generating sufficient sensitivity to measure theses signals, especially in living animals. Problems associated with the modulations in the cellular microenvironment are also tricky, but may be minimized by expressing few copies and weighing the sensitivity. The continuing development of new high-sensitivity instruments with tomographic imaging capabilities and improved spatial resolutions will play an important role in expanding the applications of bioluminescent reporters and exploiting their unique ability to precisely image the sub-cellular processes in their native microenvironment.
