Sung Bae Kim

Advances in fluorescence and bioluminescence imaging.

■ CONTENTS Fluorescence Imaging C Fluorescent Molecules for Bioimaging C Genetically Encoded Fluorescent Molecules C Synthetic Organic Fluorescent Molecules for Bioimaging F Fluorescent Nanoparticles for Bioimaging Studies F Fluorescent Probes for Biological Events G Probes for RNAs G Probes for Kinase Activity H Probes for Ions, Small Molecules, and Intracellular Environments H Bioluminescence Imaging and Quantitative Analysis K Luciferases and Substrates K Novel Luciferases K Substrates for Luciferases L Bioluminescent Probes L Probes for Protein−Protein Interactions L Probes for Small Molecules M Probes for Enzymatic Activity N Probes for Nucleic Acids N Probes for Gene Expression N Imaging of Living Subjects N Imaging of Disease Progression N Imaging of Stem Cells and Organs O Imaging of Cancers and Their Metastasis O Perspective P Author Information P Corresponding Author P Notes P Biographies P Acknowledgments Q References Q

Superluminescent Variants of Marine Luciferases for Bioassays

In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases.

A Bioluminescent Probe for Salivary Cortisol

Cortisol is a classical biomarker for the stress levels of human beings. We fabricated highly sensitive bioluminescent probes for salivary cortisol. The following strategies were contrived in the molecular design. Gaussia princeps luciferase (GLuc) was dissected into two fragments, between which an N-terminal-extended ligand binding domain of glucocorticoid receptor (GR HLBD), named Simgr4, was inserted. First, this unique single-chain probe was then situated downstream of a glucocorticoid response element (GRE) promoter in a reporter-gene system for constructing two ON-OFF switches for cortisol. Second, a circularly permutated (CP) variant of Simgr4 was formulated. The reporter-gene system exerted an improved signal-to-background (S/B) ratio of 8.5 to cortisol. Furthermore, a circularly permutated (CP) variant of Simgr4 exerted a 10× enhanced detection limit to cortisol and a long dynamic range from 10(-9) to 10(-6) M cortisol, covering all of the normal clinical ranges of serum, urine, and saliva. This optimized probe successfully determined daily fluctuations of salivary cortisol and the correlations with those by ELISA. This study is the first to investigate the contribution of the HLBD of a nuclear receptor and multiple ON-OFF switches for molecular probes and salivary cortisols.

Labor-effective manipulation of marine and beetle luciferases for bioassays

Engineering of luciferases with designed properties and functionalities collects great interest in bioassays. However, such an engineering including mutagenesis accompanies great consumption of time-and-labor. Here, I review an empirical approach to efficiently manipulate marine and beetle luciferases for bioassays, where a putative active site of luciferases is initially assigned with an in silico analysis, prior to the practical engineering, e.g. a hydrophilicity search reveals a characteristic hydrophilic region of luciferases as an engineering target. Amino acids in the hydrophilic region are recommended for a mutagenesis target to generate superluminescent variants of marine luciferases with prolonged bioluminescence. Empirical data suggest that a consecutive fragmentation to the assigned hydrophilic site greatly reduces time-and-labors on construction of single-chain probes. This review summarizes how to relieve the efforts for fabricating single-chain probes and potent variants of luciferases with excellent optical properties.

Genetically Encoded Bioluminescent Indicators for Stress Hormones

This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of corepressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP.

Bioluminescent capsules for live-cell imaging.

A bioluminescent capsule was designed for illuminating cell signaling and protein localization. The capsule consists of four components, namely, a secretion peptide (SP), a luciferase body, a cargo protein (or peptide), and a membrane-localization signal (MLS). Any functional proteins sandwiched between the luciferase body and MLS may be cartable to the plasma membrane (PM), where the capsule waits for outer signals and quickly releases the embedded luciferase in response to the signals. With this strategy of locating the capsule in the PM, the bioluminescence intensity was greatly prolonged and strengthened. A staurosporine (STS)-activated apoptosis signaling was efficiently imaged with the capsule carrying a DEVD peptide. Other functional proteins, such as fluorescent proteins and luciferases, were efficiently transported to the membrane by the capsule. A 60-nm-red-shifted bioluminescence was observed with a capsule fused with other luciferases or fluorescent proteins in living cells. This study gives a new insight regarding how to illuminate cellular signals with bioluminescence in living mammalian cells.

Intelligent Design of Nano-Scale Molecular Imaging Agents

Visual representation and quantification of biological processes at the cellular and subcellular levels within living subjects are gaining great interest in life science to address frontier issues in pathology and physiology. As intact living subjects do not emit any optical signature, visual representation usually exploits nano-scale imaging agents as the source of image contrast. Many imaging agents have been developed for this purpose, some of which exert nonspecific, passive, and physical interaction with a target. Current research interest in molecular imaging has mainly shifted to fabrication of smartly integrated, specific, and versatile agents that emit fluorescence or luminescence as an optical readout. These agents include luminescent quantum dots (QDs), biofunctional antibodies, and multifunctional nanoparticles. Furthermore, genetically encoded nano-imaging agents embedding fluorescent proteins or luciferases are now gaining popularity. These agents are generated by integrative design of the components, such as luciferase, flexible linker, and receptor to exert a specific on–off switching in the complex context of living subjects. In the present review, we provide an overview of the basic concepts, smart design, and practical contribution of recent nano-scale imaging agents, especially with respect to genetically encoded imaging agents.

Fabrication of Bioluminescent Capsules and Live-Cell Imaging

The plasma membrane of living cells is an interface of material transfers and an antenna for outer signals. This chapter provides a guide on how to fabricate bioluminescent capsules for illuminating intracellular signaling and cargo protein delivery. The capsule consists of four components, which are, in consecutive order: a secretion peptide (SP), a host luciferase body (leader), a guest protein or peptide (cargo), and a membrane localization signal (MLS). Any guest protein, including a luciferase or a fluorescent protein, may be sandwiched between the host luciferase body and MLS and may be deliverable to the plasma membrane (PM), where the capsule waits for outer signals and to quickly release the embedded luciferase in response to a specific signal. The present strategy provides an efficient molecular vehicle for cargo proteins and imaging of intracellular molecular events in living cells without substrate-derived demerits of luciferases.