Current advanced bioluminescence technology in drug discovery

Review

10.1517/17460440902804372 © 2009 Informa UK Ltd ISSN 1746-0441 373All rights reserved: reproduction in whole or in part not permitted

CurrentadvancedbioluminescencetechnologyindrugdiscoveryHideto Hoshino1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

Background:Bioluminescence technology is based on the luciferin–luciferase reaction and is generally well known as a reporter gene assay system that uses firefly luciferase. It has revolutionized the field of transcriptional analysis owing to its usability and quantitative capability. Several methods for transcription analysis have emerged in the past two decades. Recently, novel bioluminescence techniques that differ from typical approaches were developed for the detection of transcriptional regulation or direct protein–protein interactions. Objective: As each method has its own characteristics, this review summarizes the latest bioluminescence methods that are appli-cable to the field of drug discovery research. Methods: Considering the diversity of related techniques, this review covers several aspects that have been divided into the following classes: variation of reporter gene assays, secretion properties, protein–protein interaction assays in living cells and bioluminescence imaging of living cells. Results/conclusions: The practical application of several luciferins and/or luciferases and the generation of novel applications by incorporating fluorescent molecules into bioluminescence techniques will become increasingly important because bioluminescence technology has a significant potential depending on how we use it.

Keywords: BAF, beetle luciferin, BiFC assay, bioluminescence, bioluminescent imaging, BRET, celenterazine, firefly, FRET, GFP, luciferase, luciferin, multi-color, non-secretory luciferase, PCAs, reporter gene, secretory luciferase, SRET

Expert Opin. Drug Discov. (2009) 4(4):373-389

1. Introduction

Studies of luciferase were accomplished in the past, and the first beetle luciferase (BLuc) gene to be cloned and sequenced is that from the North American firefly, Photinus pyralis [1]. Its use as a reporter gene in transcriptional regulation analysis started in the late 1980s [2]. Luciferase is superior to other reporter enzymes in terms of quantitative performance and ease of measurement [3]. The most popular reporter enzyme, firefly luciferase (FLuc), was generally used at first, but the emergence of many luminous organisms containing different luciferases has initiated the cloning of luciferase genes from them [1,4-22]. New analytical systems that use several characteristics of luciferase, such as the specificity of the luciferin–luciferase reaction and the emission color, were developed. Today, biolumines-cence technology is not only a tool for transcriptional regulation analysis but is also a new approach for the detection of protein–protein interactions in a cell, or for live cell imaging.

In his recent publication [23], Shimomura states, “Harvey (1916) used the term ‘bioluminescence,’ luminescence from living organisms, possibly for the first time.” The term ‘luciferase’ pertains to bioluminescent enzymes in general and includes various luminescent enzymes. Those enzymes are isolated from luminous organisms, identified and classified on the basis of such characteristics

1. Introduction

2. Reporter gene assay

3. Secretory luciferase

4. Direct protein–protein

interaction assay in living cells

5. Bioluminescence imaging

of living cells with novel

‘artificial luciferase’

6. Expert opinion

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374 ExpertOpin.DrugDiscov.(2009) 4(4)

as ATP dependence, category of bioluminescent substrate (i.e., luciferin) and secretion property.

Luciferin–luciferase reactions are classified into two major groups according to their reaction modes, namely, ATP-dependent and ATP-independent. BLucs including FLuc are ATP-dependent enzymes. Beetle bioluminescence is characterized by a high quantum yield and a wide range of colors: green–yellow in fireflies, green–orange in click beetles and green–red in railroad worms. The variety of col-ors and the efficiency of the reactions are dependent on the protein structure of luciferase, because beetle luciferin is the common substrate for the bioluminescence reaction in all beetles. The chemical process by which luminous beetles emit light through a bioluminescence reaction consists of three successive reactions. First, BLuc catalyzes the forma-tion of luciferyl adenylate (LH2-AMP) from the substrate luciferin (LH2) in the presence of ATP (Equation 1). Then, LH2-AMP is oxidized by molecular oxygen to yield excited-state oxyluciferin and CO2(Equation 2). The excited-state oxyluciferin relaxes to its ground state with the production of yellowish–green light and AMP (Equation 3) [24,25].

(1)

BLuc + LH + ATP + Mg BLuc + LH22+

2→ -AMP + PPi + Mg2+

(2)

BLuc + LH + AMP + O BLuc - AMP - Oxyluciferin * + CO2 2 2→

(3)

n→BLuc - AMP - Oxyluciferin* BLuc + AMP + Oxyluciferin + h

All applications of the BLuc system are based on the chemical reaction theory. In fact, the emission intensity is dependent on the amounts of ATP, luciferin and luciferase; if sufficient amounts of luciferin and luciferase are available, the amount of ATP would be the determining factor of the bioluminescence intensity. This principle had already been used for ATP measurement in cells [26] and can be applied to a BLuc system for the detection of bacteria in sterile conditions [27] because bacteria contain ATP as an energy source.

With some exceptions, other non-BLucs participate in ATP-independent, simple monooxygenase reactions. For example, Renilla luciferase (RLuc) can be classified as a simple oxygenase as O2 is apparently incorporated into one of the products, oxyluciferin, whereas the second product, CO2, is derived from luciferin. The product of the reaction is an electronic excited state of the luciferase–oxyluciferin monoanion complex, which relaxes to its ground state with the production of blue light ((max = 480 nm).

(4)2 2RLuc + LH + O RLuc - Oxyluciferin *→

(5)n→ +2RLuc - Oxyluciferin* RLuc + Oxyluciferin + CO h

2. Reportergeneassay

New information regarding the genetic transcription initiation site has been accumulated in sequence through the large-scale analysis of the conventional genome and cDNA, and the analysis of transcription control regions based on them has been promoted intensively. Reporter enzyme assays have been used to estimate the interaction between promoters and/or cis-elements and transcription factors, intracellular signaling and other cellular activities, and to screen drugs both in vitro and in vivo.

Instead of β-galactosidase gene or chloramphenicol acetyl transferase gene, FLuc was widely used in transcriptional promoter analysis. Because of the wide quantitative dynamic range and the ease of use with a luminometer, the utilization of bioluminescence sparked a revolution in transcriptional assay. However, in single luciferase assay, -galactosidase enzyme was mainly used as the internal control for transfection efficiency in transient reporter gene analysis (Figure 1, top).

Later, RLuc, as an internal control, was used in combination with FLuc (Figure 1, middle) [28]. The bioluminescent substrate of RLuc is coelenterate luciferin, ‘coelenterazine,’ which differs from beetle luciferin, the substrate of FLuc. Here, the term ‘luciferin’ is the generic name for the bioluminescent substrate of luciferase; that is, luciferases belonging to different categories require different luciferin substrates. The dual luciferase system improved the convenience and accuracy of experiments, and enabled the simultaneous expression and measurement of two individual reporter enzymes in a single system.

However, even with this system, we could not monitor the expression of two or more genes simultaneously, because one of the two reporter luciferases must be used as the internal control to minimize experimental variability. To overcome the limitations of this system, we need more luciferases and a novel measurement technique that would allow us to observe the expression of two or more genes at once. Such a reporter gene assay system would be useful for the investigation of transcriptional regulation cascades that form the molecular bases of various biological functions.

Recently, Nakajima et al. developed a tricolor in vitro reporter assay system in which the expression of three genes can be monitored simultaneously by splitting the emissions from green-, orange- and red-emitting luciferases (stable luciferase green (SLG™), stable luciferase orange (SLO™) and stable luciferase red (SLR™), respectively) with long-pass optical filters (Figure 2) [29,30]. These luciferases were isolated from Brazilian railroad worm [6] and Japanese lumi-nous beetle (Table 1) [21,22] and were mutated or optimized for use in mammalian cells. Indeed, in this assay system, a

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ExpertOpin.DrugDiscov.(2009) 4(4) 375

mixed spectrum from the tricolor-emitting luciferases is observed (Figure 2A, B) and, therefore, the measured luciferase activities can be resolved into each luciferase com-ponent (Figure 2C–E). The respective activities are calculated using the simultaneous equation,

(6)

56 56 56

60 60 60

0 1 1 1

1 ,

2O O O

R R R

F G

F G O R O

G O RF R

k k kk k k

=

where G, O and R are green, orange and red luciferase activities, respectively; F0 is total relative light units measured in the absence of optical filters; F1 and F2 are the relative light units that pass through 560 nm (O56) and 600 nm (O60) long-pass filters, respectively; kGO56, kOO56 and kRO56 are the transmission coefficients of green, orange and red luciferases of the O56 filter, respectively; and kGR60, kOR60 and kRR60 are the transmission coefficients of green, orange and red luciferases of the R60 filter, respectively [30]. Emission splitting using long-pass filters is expected to minimize emission loss incurred when band-pass filters that collect a narrow range of emissions are used.

This system allows us to monitor the expression of three genes easily and simultaneously using a single luminescent

substrate in a single tube. Consequently, one is able to monitor several drug-induced transcriptional responses in a single experiment. The combined use of these multi-color luciferases and RLuc will lead to high content analyses in many research fields (Figure 1, bottom).

In the past few years, long-term live cell measurement of luminescence from luciferase has also been performed in circadian rhythm research using such specialized equipment as LumiCycle™ (Actimetrics, Wilmette, IL, USA) [31-33] and Kronos™ (ATTO Co., Tokyo, Japan) [29,34]. For example, Noguchi et al. recently reported the simultaneous monitoring of independent gene expression patterns in two lines of co-cultured live fibroblasts using green- and red-emitting beetle luciferases (SLG and SLR) through successive observation for 5 days with Kronos. The Kronos is a dish-type luminometer that provides real-time periodic luminometry having a ‘color separation’ function (as an automatic filter switching system) to simultaneously measure several luminescence signals (Figure 2F) [34]. Unlike the conventional in vitro analysis of cell lysate, the apparatus will enable us to perform not only high- throughput but also high-content luminometry of self- luminous cells expressing luciferase or self-luminous tissues, such as those from transgenic animals containing the luciferase gene.

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Figure1.Advances in reportergeneassaysystem. Schematic showing the transition of the measurement method of luciferase activity. A. Single luciferase assay system from 1988 (first generation). B. Dual luciferase assay system from 1996 (second generation). C. 3 + 1-color luciferase assay system from 2005 (third generation).I.C.: Internal control; SLG: Stable luciferase green; SLO: Stable luciferase orange; SLR: Stable luciferase red.



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Figure2.Multi-colorluciferaseassaysystem.A. Bioluminescence spectra of green (SLG, green line), orange (SLO, orange line) and red (SLR, red line) luciferases, and the transmission spectra of 560- (O56, black solid line) and 600-nm (R60, black dotted line) long-pass filters. Photograph (inset) shows the bioluminescence of silkworm expressing SLG (left), SLO (middle) and SLR (right) luciferases, after mixing luciferin. B. Mixed bioluminescence spectrum of SLG, SLO and SLR (black line) and the transmission spectra of O56 (orange line) and R60 (red line) filters. C–E. Measurement of luciferase activities using long-pass filters. The simultaneous equation for calculating luciferase activity is shown at the bottom of the figure [30]. F. The 35 mm culture-dish-based multi-color luminometer is capable of separating several luminescence signals by automatically switching filters. First, total luminescence is measured without any filters, and then luminescence is measured with an optical filter (F1O56 and F2R60). Based on the measurements made with the filter and the filter transmittance for each luminescence component that was measured beforehand, the luminescence of each signal is calculated. Figure2A–E was constructed on the basis of Nakajima’s paper [30].SLG: Stable luciferase green; SLO: Stable luciferase orange; SLR: Stable luciferase red.

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3. Secretoryluciferase

Several luciferases, such as FLuc and RLuc, are categorized as non-secretory luciferases. Recently, light blue luminescent secretory luciferases, such as Gaussia luciferase (GLuc; max = 480 nm), Vargulla luciferase ((max = 460 nm), and Cypridina luciferase (CLuc; max = 460 nm), were isolated and identified from luminous marine organisms [8,9,20,35]. Secretory luciferases have a distinct advantage over non- secretory ones; that is, the luciferase enzyme expressed in cells is secreted into the cell-culture medium (Figure 3A).

From a cellular secretion viewpoint, secretory luciferase is in itself used as a tool to monitor the protein secretory pathway [36,37] and to quantify autophagy [38] in living cells. For example, fibroblasts from torsion dystonia 1 patients were found to secrete markedly less GLuc compared with control fibroblasts [39]. The studies demonstrated the sensi-tivity of this reporter system for the quantitation of process-ing through the secretory pathway and supported the role of the responsible gene, torsinA, as an estrogen receptor chap-erone protein [39]. This GLuc secretory assay offers a simple and sensitive means to evaluate the integrity of the secretory pathway and to screen for drugs and agents that can facilitate protein processing in disease states.

Using both the properties of these luciferases and specialized perfusion apparatus, one can gain temporal transcriptional

information from living cells without cell lysis (Figure 3B). Yamagishi et al. reported the development of a reporter system with another secretory luciferase, CLuc, for a pharmacological assay that is based on targeted promoter activity [40]. They successfully monitored circadian oscilla-tion of Bmal promoter and demonstrated that the CLuc reporter assay in combination with perfusion culture is a suitable pharmacological tool for drug discovery.

The activities of both luciferases (i.e., GLuc and CLuc) in the culture medium can be easily quantified from the light intensity produced after reacting with their substrates. The luciferins of GLuc and CLuc are different: they are coelenterazine [35] and cypridina luciferin [41-43], respectively. Recently, Wu et al. reported a dual reporter assay that uti-lized their enzymatic properties and substrate specificities. The two bioluminescence reaction systems did not crossre-act and the half-lifes for decay were similar (> 60 h at room temperature, pers. commun.). Thus, the activities of the two secretory luciferases could be measured separately and accurately [44].

Compared with previous reporter assay systems, this dual reporter assay system has several advantages: i) it can be performed sequentially for hours to days using the same cell population; ii) the activities of the two luciferases are relatively high and can be measured in a small volume (< 10 µl medium); and iii) the procedure is very simple and

Table1.Characteristicsofvariousluciferases.

Secretiontype Luciferin Molecularsize(kDa) Emissionpeak(nm) Distributionsource Remarks

Naturalluciferase

Non-secretion

FLuc Firefly luciferin 61 562 Promega [1]

RLuc Coelenterazin 36 480 Promega [7]

CBLuc Firefly luciferin 60 613 Promega [4]

CBLuc Firefly luciferin 60 537 Promega [4]

SLR Firefly luciferin 61 630 TOYOBO [6]

SLO Firefly luciferin 60 580 TOYOBO [5]

SLG Firefly luciferin 60 550 TOYOBO [5]

Bacterial luciferase FMNH2 80 490 -

Secretion

GLuc Coelenterazine 20 480 Lux Biotechnology

CLuc Cypridina luciferin 61 460 ATTO [8]

Artificialluciferase

Non-secretion

BAF-C Coelenterazine 64 480 - Unpublished

BAF-A Coelenterazine 64 505 - [52]

BAF-G Coelenterazine 64 510 - [52]

BAF-Y Coelenterazine 64 525 - [52]

BAF-R Coelenterazine 64 572 - Unpublished

BAF: BRET-based auto-illuminated fluorescent-protein; BRET: Bioluminescence resonance energy transfer assay; CBLuc: Click beetle luciferase; CLuc: Cypridina luciferase;

FLuc: Firefly luciferase; GLuc: Gaussia luciferase; SLG: Stable luciferase green; SLO: Stable luciferase orange; SLR: Stable luciferase red; RLuc: Renilla luciferase.



does not require special equipment. Thus, the dual reporter assay is suitable for high-throughput analysis of gene transcription in living cells.

In the past, the procedure involved the use of two tubes for one assay, which required accurate separation of the sample. However, Wu et al. overcame that problem excel-lently: using 0.1% sodium dodecyl sulfate that inhibited GLuc activity almost completely but not Cluc activity, they accomplished a single-tube dual reporter assay system [44]. This reporter assay system could be used to analyze in detail the transcriptome and the promoterome, as well as to screen for new drugs or to detect harmful chemicals.

It is expected that combining secretory luciferase techniques and non-secretory luciferase techniques will extend their application. Indeed, Nishide et al. performed circadian transcription analysis using FLuc and Vargulla luciferase

for cultured tissues from transgenic animals [45], and Michelini et al. demonstrated a triple reporter gene assay system that was developed in the high-throughput 96-well micro titer plate format combining two different intracellular FLuc variants as reporters and a secretory luciferase as internal control [46,47].

4. Directprotein–proteininteractionassayinlivingcells

Bioluminescence approaches for the detection of direct protein–protein interactions in mammalian cells are subdivided into three major categories: bioluminescence resonance energy transfer (BRET) assay, protein fragment complementation assay (PCA) and modified bioluminescence-related techniques. They are used to quantitatively measure real-time protein–protein

Culture chamber

Lumino-meter

Peristalticpump

Drugvial

Mediumbottle

3-waystopcock

Fractioncollector

Mammalian cellMedium

Lucifern

Siliconerubberstopper

A.

B.

Figure3.Secretory luciferaseand itsapplication.A. Secretory luciferases are secreted into extracellular space through a typical cellular secretory pathway. B. A silicon plug containing two ports inserted with PEEK tubes of 1 mm inside diameter was attached to each well of the 12-well plate. The resultant chamber was set at a height of ∼ 9 mm by using stoppers to position the plug. The ends of the two PEEK tubes were adjusted to heights of 1 – 2 mm from the bottom of the well to collect 0.5 ml of medium in the chamber during perfusion. One port of the perfusion chamber was connected to a fraction collector and another port was connected to a three-way stopcock that branched into a medium bottle with a screw cap and a vial for the drug, by using Pharmed tubes and Luer fittings. Figure3B was constructed on the basis of Yamagishi’s paper [40].

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interactions in mammalian cells or to study signal-controlled protein–protein (or peptide) interactions.

4.1 BRETassayBRET is a straightforward biophysical technique for studying protein–protein interactions in living cells. Similar to fluorescence resonance energy transfer (FRET), this method is based on the principle of resonance energy transfer between an energy donor and an energy acceptor. BRET involves non-radiative energy transfer from a donor bioluminescent enzyme to a suitable acceptor fluorescent molecule after substrate oxidation. It only takes place when several condi-tions, including distance, relative orientation, and degree of spectral overlap between donor and acceptor, are fulfilled [48]. The transfer of excited-state energy is in inverse proportion to the sixth power of the distance between an energy donor and acceptor dipoles, providing an effective range of < 10 nm [49]. This distance is comparable to the dimensions of biological macromolecular protein complexes and thus makes BRET an appropriate approach for evaluating protein interactions. Proteins of interest can be genetically fused to a bioluminescent enzyme or a fluorescent protein. Resonance energy transfer occurs when the proteins of interest bring the donor closer to the acceptor, a distance generally indicative of interaction between the proteins of interest, either directly or as part of a complex. The resulting acceptor energy emission can then be detected relative to the donor emission.

It is commonly believed that there are practical problems in FRET-based systems, including photo bleaching, auto-fluorescence and simultaneous excitation of both donor and acceptor fluorophores, and that one can circumvent those problems by using BRET. Dacres et al. compared directly the two methods based on fluorescent proteins by perform-ing cleavage experiments of a thrombin-protease-sensitive peptide sequence between donor and acceptor proteins [50]. They reasoned that BRET has higher sensitivity and accu-racy than FRET for use in proximity assays, such as protease cleavage assays or protein–protein interaction assays.

In BRET, the selection of donor, acceptor and substrate combination is of considerable importance. Generally, BRET technology uses the combination of RLuc as the energy donor, a variant of green fluorescent protein (GFP) from Aequorea as the energy acceptor and coelenterazine as the substrate of the luciferase. The derivation of donor and acceptor from different species is of minor importance because the sea pansy, Renilla reniformis, has its own GFP (RrGFP), an intrinsic BRET acceptor, and RrGFP was found to be structurally similar to GFP from Aequorea, with the exception of its much stronger dimerization interface [51], and efficient BRET occurs between the GFP variant from Aequorea and RLuc [52]. The direct interaction between RLuc and GFP is highly specific as well as highly sensitive to ion strength and needs high protein concentration [7,53,54]; therefore, it does not occur under physiological cellular

conditions. Furthermore, RLuc mutations can increase emission intensity [55,56].

As the substrate of RLuc, coelenterazines are small hydro-phobic compounds that readily permeate the cell membrane, thereby permitting analysis of intact cells. The donor spectrum is dependent on the type of coelenterazine used (coelenterazine h: max = 480 nm, bisdeoxycoelenterazine: max = 395 nm, inset of Figure 4A), in addition to the donor protein itself. The original BRET system uses RLuc as the donor, coelenterazine h as its substrate and a yellowish–green fluorescent protein (YFP) as the acceptor [57]. In the original method, RLuc and YFP emit light with a peak wavelength of ∼ 480 and 527 nm, respectively, resulting in poor spectral resolution (i.e., the difference between donor and acceptor emissions) of 47 nm. In addition, RLuc pro-duces a broad emission spectrum that substantially overlaps with the YFP emission spectrum. The RLuc emission masks the YFP emission, thereby contributing to a high YFP background and decreasing the signal-to-background ratio of the system. However, recently, bisdeoxycoelenterazine (better known as DeepBlue C™ (DBC; PerkinElmer, Waltham, Massachusetts, USA)) is mainly used in BRET2 technique. Use of DBC as the substrate results in a striking decrease of the luminescence intensity (∼ 0.2% coelenterazine) and a simultaneous wide shift of the emission peaks of the donor and the acceptor. The reaction produces light at 395 nm, which is a much shorter wavelength than that of native coelenterazine. In this BRET2 technique, GFP2 is used as the energy donor. GFP2 has an excitation spectrum that is adapted to this wavelength and, on energy absorp-tion, re-emits light at 510 nm, thus providing a broad spectral resolution between donor and acceptor emission peaks (∼ 115 nm). This broad spectral resolution permits the selection of filters that absorb the entire bandwidth of the donor and acceptor emission peaks, thereby increasing robustness of the detection.

Modified BRET techniques have emerged in recent years. Pfleger et al. presented a new derivation of BRET technology termed ‘extended BRET’, which enables real-time monitoring of protein–protein interactions for long periods using a stable coelenterazine analogue named ‘EnduRen™, [58]. Kocan et al. also demonstrated improvements to all three generations of BRET (BRET, BRET2 and extended BRET) by using such high-performance luciferases as RLuc8, a variant of RLuc containing eight amino acid substitutions [55], developed in the Gambhir laboratory [59].

4.2 PCAassayPCAs using luciferase are rapidly becoming a common tool for studies of the dynamics of protein–protein interactions in living cells. In this strategy, two proteins of interest are fused to the complementary N-terminal fragment or C-terminal fragment of a luciferase. Functional complemen-tation between the reporter protein fragments, mediated by the interaction of the two fusion proteins, results in a



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Figure 4. Various bioluminescence assays for detection of direct protein–protein interactions. Summarized schematic representations of BRET assay (A), protein fragment complementation assay (B), combined BiFC assay (C), combined BiBC assay (D) and SRET assay (E). Inset in (A) shows the emission spectra of Renilla luciferase using coelenterazine or DBC as luciferin, respectively. Note the difference of the scales of the two Y-axes.BiBC: BRET-BiFC-bimolecular bioluminescence complementation assay; BiFC: BRET-bimolecular fluorescence complementation assay; BRET: Bioluminescence resonance

energy transfer assay; DBC: DeepBlueC; RET: Resonance energy transfer; SRET: Sequential RET assay.

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Figure 4. Various bioluminescence assays for detection of direct protein–protein interactions. Summarized schematic representations of BRET assay (A), protein fragment complementation assay (B), combined BiFC assay (C), combined BiBC assay (D) and SRET assay (E). Inset in (A) shows the emission spectra of Renilla luciferase using coelenterazine or DBC as luciferin, respectively. Note the difference of the scales of the two Y-axes (continued).BiBC: BRET-BiFC-bimolecular bioluminescence complementation assay; BiFC: BRET-bimolecular fluorescence complementation assay; BRET: Bioluminescence resonance




quantifiable signal that can be used to investigate the localization and regulation of protein interactions in their normal environment (Figure 4B).

Ozawa et al. demonstrated that FLuc can be split at amino acid positions 437 and 438 and used with DnaE intein (a catalytic subunit of DNA polymerase III) in a ‘reconstitution’ strategy to detect the insulin-induced inter-action of phosphorylated IRS-1 with its target N-terminal SH2 domain derived from phosphatidylinositol 3-kinase in a cell culture assay [60]. Paulmurugan et al. subsequently reported the possibility of splitting FLuc and of using split reporter ‘complementation’ without protein splicing [61]. They also showed that the complementation method is as sensitive as the intensin-mediated reconstitution method under their experi-mental conditions. In the past, bioluminescent reporter enzymes, such as click beetle luciferase [62], RLuc [63] and Gluc [64], have been adopted for split protein strategies by finding various split sites for each luciferase, namely, click beetle luciferase: 439 – 440; RLuc: 229 – 230; and GLuc: 93 – 94.

4.3 Modifiedbioluminescence-relatedtechniquesThe identification of higher order oligomers in the plasma membrane is essential to decode the properties of molecular

networks controlling intercellular communication. Hu et al. reported initially the application of PCA to fluorescent pro-teins as “bimolecular fluorescence complementation (BiFC) assay” [65,66]. Recently, several research groups have reported the utility of combining BRET and BiFC assays [67-71]. The dual BRET/BiFC (or combined BRET-BiFC) assay enables detection of oligomerization of the proteins of interest in living cells; initial protein interactions induce the comple-mentation of fluorescence acceptor and subsequent interactions prompt BRET between the bioluminescent donor and the functionally reconstituted fluorescent acceptor (Figure 4C). Heroux et al., for instance, combined BiFC with BRET to probe the stoichiometric arrangement of the calcitonin gene-related peptide receptor complex [69]. The combined BRET-BiFC assay revealed that functional calcitonin gene-related peptide recep-tors are a result of the association of a homo-oligomer of the calcitonin receptor-like receptor with a monomer of the acces-sory protein receptor activity-modifying protein-1. In addition to demonstrating the existence of an unexpected asymmetric oligomeric organization of a G-protein-coupled receptor (GPCR), their study illustrated the usefulness of the dual BRET/BiFC assay as a powerful tool for analyzing constitutive and dynamically regulated multi protein complexes.

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Figure 4. Various bioluminescence assays for detection of direct protein–protein interactions. Summarized schematic representations of BRET assay (A), protein fragment complementation assay (B), combined BiFC assay (C), combined BiBC assay (D) and SRET assay (E). Inset in (A) shows the emission spectra of Renilla luciferase using coelenterazine or DBC as luciferin, respectively. Note the difference of the scales of the two Y-axes (continued).BiBC: BRET-BiFC-bimolecular bioluminescence complementation assay; BiFC: BRET-bimolecular fluorescence complementation assay; BRET: Bioluminescence resonance


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In a very recent published work, Carriba et al. combined BRET and FRET in a technique called ‘sequential BRET-FRET (SRET)’ that permits identification of heteromers formed by three different proteins [72]. In SRET, the oxidation of an RLuc substrate, coelenterazine, by an RLuc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. They described two variations of SRET that use different RLuc substrates (i.e., native coelenterazine and DBC) with appropriately paired acceptor fluorescent proteins. Using SRET, they identified complexes of cannabinoid CB1, dopamine D2 and adenosine A2A receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level. SRET is expected to circumvent several practical problems of existing FRET techniques as described above.

4.4 PracticalapplicationsofbioluminescencetechniquesThe bioluminescence techniques (i.e., BRET assay, PCA, BRET-BiFC assay and SRET assay) mentioned previously adopt essentially the same approach for the detection of direct protein–protein interactions in living cells. On the other hand, each protein of interest, in itself, possesses an intrinsic structure of its own; therefore, the success or failure of an intended assay system depends on the intrinsic struc-ture or the interaction mode. Such a diversity of approaches would expand the possibilities for the construction of individual assay systems.

For example, GPCRs and the surrounding molecules are major targets of direct protein interaction assays using bioluminescence. GPCRs interact with a range of proteins either directly or indirectly to function as agonist-modulated signaling complexes and play an extremely important role in transducing extracellular signals across the cell membrane with high specificity and sensitivity. They are central to many endocrine systems and neurotransmitter pathways, and are also major pharmacological targets. BRET tech-niques have been used to detect ligand-dependent homo- and hetero-oligomerization of GPCRs in living cells. Using BRET, Kroeger et al. were also able to confirm the differen-tial ability of the thyrotropin-releasing hormone receptor (TRHR) and the gonadotropin-releasing hormone receptor to interact with -arrestin. The development of biophysical techniques to study receptor–protein interactions in living cells provides an effective means to obtain direct evidence of the role of these events in GPCR function [73]. Hanyaloglu et al. demonstrated that TRHR subtypes are differentially regulated by -arrestins and also provided the first evidence that the interactions of TRHRs with -arrestin may be altered by hetero-oligomer formation [74]. BRET has demonstrated GPCR interactions with G protein [75], β-arrestins [73,74,76], adenylyl cyclase and a subunit of an inwardly rectifying K+ channel [68,77]. Perroy et al. developed

an interesting BRET assay system to measure the ubiquitination of -arrestin 2, a regulatory protein implicated in the modulation of GPCRs [78]. It is a useful tool to study the real-time dynamic ubiquitination of proteins without perturbing the physiological environment of cells and to understand cellular functions regulated by post-translational events [79].

Bioluminescence methods are used to investigate not only direct protein–protein interactions but also drug-mediated protein–protein interactions. Michelini et al. developed in vitro and in vivo monitoring systems for estrogen-like compounds using BRET [80]. The assay is based on the direct evaluation of estrogen receptor homodimerization as a result of estrogen-like compound binding and allows for the evaluation of the estrogen-like activity and synthetic xenoe-strogens from biological and environmental samples. On the other hand, Paulmurugan et al. developed the PCA to esti-mate small molecule-mediated interactions of proteins [81-83]. They evaluated the heterodimerization of human proteins FRB and FKBP12 mediated by the small molecule rapamy-cin and indicated the possibility that the reporter system can be used to efficiently screen for small molecule drugs that modulate protein–protein interactions and to assess drugs in living animals. Similarly, several research groups have reported split-complementation approaches for the assessment of interactions between sex hormones and corresponding nuclear receptors [62,84] and for the detection of specific RNAs [85].

From a subcellular localization standpoint, Kim et al. developed bioluminescent split-complementation assay systems to monitor nuclear trafficking of androgen receptor (AR) [86], glucocorticoid receptor [87], signal transducer and activator of transcription 3, and sterol-regulatory element binding protein-2 [88] in vitro and/or in vivo. They demon-strated quantitative cell-based sensing of ligand-induced nuclear translocation of AR and glucocorticoid receptor or of phosphorylation- and proteolysis-induced nuclear trans-port of signal transducer and activator of transcription 3 and sterol-regulatory element binding protein-2, respectively. Kanno et al. developed a bioluminescence indicator to monitor the release of apoptosis-related proteins from the mitochondria in living cells [89]. Furthermore, the indicators enabled noninvasive in vivo imaging of AR translocation in mouse brains and of Smac/DIABLO protein release in living mice with a charge-coupled device imaging system [86,89]. These rapid and quantitative analyses performed in vitro and/or in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

5. Bioluminescenceimagingoflivingcellswithnovel‘artificialluciferase’

In the past, FLuc was mainly used for bioluminescence imaging at the live cell level. The emission spectra of FLuc,



however, are decomposed into one pH-sensitive (peaking at around 560 nm) and two pH-insensitive (peaking at around 620 and 670 nm, respectively) Gaussian components, and as pH decreases, the quantum yield decreases rapidly [90]. Indeed, a weak red emission is dominant in the spectrum of FLuc measured in living cells; the emission in living cells is not intense compared to that in pH-optimized in vitro analysis. On this account, bioluminescence imaging with FLuc requires long-time exposure for 30 min but both temporal and spatial resolutions at the single-cell level were not so high [91].

In nature, BRET is an intrinsic mechanism possessed by such sea organisms as jellyfish and sea pansy to induce GFP fluorescence through bioluminescence reactions [92]. Recently, we reported BRET-based bioluminescence imaging with high spatiotemporal resolutions [52]. This is the first example that makes maximum use of the advantages of BRET as a means to excite fluorescent proteins for single-cell imaging. We developed simple fusion proteins consisting of a GFP variant and an RLuc and named these bioluminescent probes ‘BRET-based auto-illuminated fluorescent proteins (BAFs).’ As illustrated in Figure 5A, BAF is a fluorescent protein accompanied with luciferase as an intramolecular excitation light source. BAF forms a new category called ‘artificial luciferase.’ The addition of luciferin leads to the induction of fluorescence from the fluorescent protein in the BAF probe.

Generally, longer wavelength light should have higher light transmission properties in deep body and is preferable for whole animal body imaging, as blue wavelength is attenuated in biological tissues. Recently, Loening et al. developed several high-performance red-shifted RLuc vari-ants for small-animal imaging [56]. They presented an oppor-tunity for us to develop new BAF variants. As Miyawaki predicted in his review [93], BAF is broadly applicable to other fluorescent proteins; indeed, we have developed several red-shifted BAF probes (our unpublished data). As shown in Figure 5B, oxidation of coelenterazine h by RLuc or its variants in several BAF probes led to viewable luminescent colors from blue to orange.

Interestingly, BAF does not impair the original luciferase activity by protein fusion in the least. BRET, in contrast, enhances the emission intensity of the original RLuc [52]. For example, in the case of BAF on enhanced yellow fluorescent protein (i.e., BAF-Y), BAF-Y exhibited a sevenfold increase in emission intensity relative to RLuc. Moreover, when RLuc variants with enhanced emission intensity, such as RLuc8 [55], were used in BAF-Y, the eventual BAF-Y variant (named enhanced BAF-Y; eBAF-Y) exhibited greater than 25-fold enhancement in lumine-scence intensity compared to the original RLuc. Therefore, eBAF-Y is one of the most powerful bioluminescent probes. If combined with the high-performance CCD detector system shown in Figure 5C, we can perform the once-unthinkable real-time observation of cellular activities

using only bioluminescence by adding coelenterazine into the culture medium (Figure 5D–I).

Noteworthy is that despite an apparent molecular mass of 64 kDa, when fused as subcellular localization signals or proteins at the N terminus or C terminus of (e)BAF-Y, they can be also used as bioluminescent tags, as are many GFP variants (Figure 5E–H and our unpublished data). One characteristic of bioluminescence is its extremely low emission background that results in the high S/N ratio of images. Indeed, BAF-Y realizes high-contrast imaging. We recently obtained more than three frames of detailed cell images per minute; consequently, we can visualize the apoptosis process in detail (Figure 5I).

6. Expertopinion

Approximately 20 years have passed since FLuc was initially put to practical use in life science research as an innovative technique, and improvements of reporter gene technologies, as are well known, have been effected time and time again. However, the existing technologies do not always give the best performance. For example, FLuc shifts the spectrum from yellowish green to red in response to pH and does not emit as high an intensity of light in living cells as expected from the optimized in vitro assay, because the local pH conditions in cultured cells may be not optimal for the emission of FLuc. There is still room for the development of bioluminescence technol-ogy. If a new luciferase that exhibits increased spectral pH-independency were developed, it would become a powerful tool in in vivo analysis.

Many attempts to increase the color range of bioluminescence emissions have been made and several have succeeded: the combined utilization of luciferases requiring different luciferins [28], the mutagenesis of luciferase [30,56,94] and the cloning of novel luciferases from different luminescent bee-tles with the identical luciferin [6,19]. The emission of several colors from luciferases is expected to increase the amount of information obtained at one time. It is worth noting that we are now able to co-instantaneously and quantitatively moni-tor several gene expressions for a long time. These multi-color luciferase technologies and their related applications might lead to not only high-throughput analysis but also to high content analysis for new drug discovery or for various toxicity assessments, namely, immunotoxicity, neurotoxicity and teratogenicity, of chemicals. By combining several types of luciferases, one will be able to gain much intracellular information depending on one’s ideas.

Fluorescence techniques using GFP have flourished for more than a dozen years and are essential in almost all bioscience research areas, as reflected by the 2008 Nobel Prize in Chemistry:“Glowing proteins: a guiding star for biochemistry.” On the other hand, bioluminescence tech-niques undoubtedly are superior to all existing fluorescence methods in terms of quantitative performance, owing to the

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Figure5.Bioluminescencesingle-cellimagingusingBAFprobe.A. Conceptual diagram of BAF. B. Invitro actual emission color variations of several recombinant BAF proteins purified from Escherichiacoli BL-21 strain. C. An example of a bioluminescence imaging apparatus. D–F. Bioluminescence images of cells expressing BAF-Y, nuclear-targeted NLS-BAF-Y or cytoplasm-targeted NLS-BAF-Y-NES, respectively. Gand H. Bioluminescence images of cells expressing histone H2AX-eBAF-Y or eBAF-Y-histone H2AX fusion proteins. Note that eBAF-Y functions as a bioluminescent tag in the same way as green fluorescent protein variants do in the fluorescence technique. I. Bioluminescence sequential images of the apoptotic process of a cell expressing eBAF-Y. Numbers at the lower right of the each photograph represent observation time (min).BAF: BRET-based auto-illuminated fluorescent-protein; BAF-Y: BAF on enhanced YFP; BRET: Bioluminescence resonance energy transfer assay; eBAF-Y: enhanced

BAF-Y; NES: Nuclear export signal; NLS: Nuclear localization signal; YFP: Yellow green fluorescent protein.



extremely low luminescence background and the wide dynamic range. We tend to think that fluorescence techniques are the competitors of bioluminescence methods; however, they do not necessarily compete with each other. Rather, we should use them in a complementary manner so that they would be able to make up for each other’s deficiencies.

In particular, the development of novel bioluminescence methods has been remarkable over the past several years. The utilization of fluorescent proteins in bioluminescence methods, such as BRET, BRET-BiFC and SRET assays, might be one of the solutions to the practical problems in FRET methods, such as photo bleaching, auto-fluores-cence and simultaneous excitation of both donor and acceptor fluorophores. In fact, BRET methods have revealed the molecular behaviors of GPCRs and related reg-ulatory proteins in living cells, and BRET-BiFC and SRET methods have demonstrated hitherto undetected higher order homo- and/or hetero-oligomelization of cell membrane receptors in intact cells. These novel biolu-minescent protein interaction assays are very useful for understanding the properties of molecular networks regulating intracellular communication.

The utility of bioluminescence in cellular imaging seems to be undervalued because of its low emission intensity. As seen in natural BRET phenomenon, however, some luminous marine organisms, such as jellyfish or sea pansy, use both bioluminescent enzyme and GFP by synchronizing them for their own natural ‘whole body imaging.’ The combination of high-performance bioluminescent probes and high-end detection apparatuses is expected to expand the utility of bioluminescence in practical applications achieved only by existing fluorescence techniques so far. Our BAF technique is a straightforward example as it has

opened the door to real-time bioluminescence imaging at the single-cell level. In addition, as shown in Figure 5B, we have already developed multi-color BAF variants and are aiming for the realization of multi-color bioluminescence imaging at the single-cell level. Several quantum-dot conjugated luciferases have emerged and are used in noninvasive small-animal imaging [95-97]. Bioluminescence imaging can bypass the risk of photocytotoxicity derived from strong excitation light and can provide real-time observations for many hours and quantitative interpretation of various in vivo phenomena. Therefore, it merits further research. It has become extremely important to develop novel approaches by incorporating fluorescent proteins or fluores-cent materials into bioluminescence techniques.

Finally, there are still many luminescent organisms existing in nature and some of them have emission processes (luciferin form, ATP-dependent and so on) that differ from those of known luminescent organisms. In fact, the detailed luminescence mechanisms of several organisms, such as fire-fly squid and arachnocampa, remain unexplained. Resolu-tion of detailed processes in the future would lead to new variations in bioluminescence technology. Bioluminescence technology in itself still has tremendous potential.

Acknowledgement

The author thanks Dr Yoshihiro Nakajima and Dr Chun Wu (AIST) for discussion and valuable advice.

Declarationofinterest

The author states no conflict of interest and has received no payment in preparation of this manuscript.

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AffiliationHideto Hoshino PhDCell Dynamics Research Group Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka, 563-8577, Japan Tel: +81 72 751 7997; Fax: +81 72 751 9628; E-mail: [email protected]

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Current advanced bioluminescence technology in drug discovery