Recent progress in design of protein-based fluorescent biosensors and their cellular applications.

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Review

Recent progress in design of protein-based fluorescent biosensors and their cellular applications Tomonori Tamura, and Itaru Hamachi ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 15 Oct 2014 Downloaded from http://pubs.acs.org on October 16, 2014

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ACS Chemical Biology

Table of contents 66x39mm (300 x 300 DPI)

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ACS Chemical biology

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Review: Recent progress in design of protein-based

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fluorescent biosensors and their cellular applications

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4 Tomonori Tamura1and Itaru Hamachi1,2*

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Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto

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University, Katsura, Kyoto 615-8510, Japan. 2

Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.

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Email: [email protected]

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*Corresponding author: Itaru Hamachi, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Kyoto

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615-8510, Japan. Tel: 075-383-2757; Fax: 075-383-2759; Email: [email protected]

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ABSTRACT

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Protein-based fluorescent biosensors have emerged as key bio-analytical tools to visualize and

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quantify a wide range of biological substances and events in vitro, in cells, and even in vivo. On the

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basis of the construction method, the protein-based fluorescent biosensors can be principally classified

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into two classes: 1) genetically encoded fluorescent biosensors harnessing fluorescent proteins (FPs),

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and 2) semisynthetic biosensors comprised of protein scaffolds and synthetic fluorophores. Recent

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advances in protein engineering and chemical biology not only allowed the further optimization of

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conventional biosensors, but also facilitated the creation of novel biosensors based on unique strategies.

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In this review, we survey the recent studies in the development and improvement of protein-based

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fluorescent biosensors, and highlight the successful applications to live cell and in vivo imaging.

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Furthermore, we provide perspectives on possible future directions of the technique.

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MAIN TEXT

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1. Introduction

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The last two decades have witnessed tremendous progress in fluorescent biosensors that are defined

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as integrated biomolecules (typically nucleic acids or proteins) capable of reporting a molecular

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recognition event as a fluorescent signal.1 In particular, protein-based fluorescent biosensors have

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revolutionized many areas of biological science because they allow imaging and quantitative

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measurement of specific biomolecules and events in cells, as well as in vitro.1-7 The biosensor generally

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consists of a protein scaffold as a recognition module for the target analyte and a fluorescent transducer

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to convert the interaction between the protein and the analyte into a fluorescent signal change in its

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intensity or wavelength. Compared to other biomolecules, the excellent ability to recognize various

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biologically relevant molecules with high specificity and affinity is one of the major advantages in

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proteins, which permits the construction of robust biosensors toward targets of high diversity. In

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addition, protein-based biosensors are now considered as powerful platforms for high-throughput

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screening (HTS) for drug candidates using pharmacologically attractive proteins as a recognition

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module.8,9

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Protein-based fluorescent biosensors can be divided into two classes depending on the type of

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fluorescent transducers: 5,6 1) genetically encodable fluorescent biosensors utilizing fluorescent proteins

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(FPs), 2) semisynthetic fluorescent biosensors that are constructed by site-specific chemical

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modification of protein scaffold with a synthetic fluorescent dye. In both classes, the creation of a novel

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fluorescent biosensor for a given target is still not straightforward due to the lack of general design

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principles to transduce an analyte-binding process into a fluorescent signal change. Nevertheless,

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increasing demands for live-cell imaging have driven researchers to develop a variety of new strategies

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for the sensor design and optimization. Such efforts provide not only unprecedented tools to elucidate

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biological systems, but also a deep insight into the future design guidelines to create biosensors more

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rationally. In this review, we briefly outline the latest design strategies for the development and


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improvement of protein-based fluorescent biosensors, and describe successful examples in their cellular

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applications.

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2. Genetically encoded fluorescent protein (FP)-based biosensors Aequorea victoria green fluorescent protein (GFP) and its homologues are most commonly used to

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visualize spatiotemporal information and dynamics of a protein of interest in live cells.3,13-15 Since the

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first discovery and cloning, the GFP family have been engineered to produce a variety of colorful

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mutants ranging from blue to red.16-18 Such rich variants expanded available strategies to be used for

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creating genetically encoded FP-based biosensors capable of imaging and quantifying more complex

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events in living cells with high spatial and temporal resolution. More recently, several FPs composed of

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protein scaffolds other than GFP derivatives, such as bacterial phytochromes19-22, rhodopsins23, and

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fatty-acid-binding protein (FABP) family24, have been adopted to biosensors construction. These new

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type of FPs with unique chemical, spectral, and biological properties are now providing the further

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diversity and flexibility of biosensor design strategies.

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FP-based biosensors have several advantages compared to synthetic dye-based biosensors.10,11 First,

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FP-based biosensors are genetically encodable, allowing the easy incorporation by transfection into cells

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where they are produced by the endogenous cellular transcriptional and translational machinery.

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Secondly, the sensors can be selectively localized to subcellular organelles by fusing specific signal

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sequences to the N- or C-terminus of the FP sensor. Finally, the concentration of sensor can be

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controlled by putting it under control of a chemically inducible promoter.

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In this section, we describe the recent strategies for construction of fluorescent biosensors relying on

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GFP-like proteins and non-GFP scaffolds. The vast number of biosensors using GFP-like proteins are

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further classified into three sub-categories4-6; 1) Förster resonance energy transfer (FRET)-based

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biosensors, 2) single FP-based biosensors, 3) bimolecular fluorescence complementation (BiFC)-based

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biosensors. Although genetically encoded bioluminescent-based sensors are also powerful especially for

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applications in tissues and animals, the scope of this review focused on only fluorescent-based ACS Paragon Plus Environment

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biosensors owing to limitation of space. More comprehensive information on fluorescent and

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bioluminescent protein-based biosensors is available in other excellent reviews.1,7,25

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2-1. FRET-based FP sensors

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FRET is a phenomenon of non-radiative energy transfer from a donor fluorophore in its excited state

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to an accepter fluorophore, which only occurs when the two fluorophores are in proximity (1-10 nm) of

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each other, and the emission spectrum of the donor overlaps with the excitation spectrum of the

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acceptor.1,2 FRET-based sensors generally consist of a recognition module and two FPs. The currently

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preferred FRET pair of FPs is cyan FP (CFP) and yellow FP (YFP) couple.26 Thanks to the

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improvement of red-FP (RFP), red-shifted FRET pairs are also utilized, together with the conventional

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CFP/YFP pair to image multiple signaling networks.26-29

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In FRET-based biosensors, the analyte-induced conformational changes can be transduced into a

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ratiometric fluorescent change through a substantial modulation of FRET efficiency between donor and

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acceptor FPs. Some of the most effective design formats are schematically represented in Figure 1a-d.

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These have been proven to be useful for generating FRET-based biosensor for various intracellular

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molecules and events (e.g. enzymes activity, metal ions, chemicals, post-translational modifications,

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protein-protein interactions (PPIs)).1-7,10-12 However, even after the iterative optimization, the signal

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change is small to moderate, in many cases, mainly due to the structural flexibility of the sensor that

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generally requires a long flexible linker for proper folding of each domain.11,12,30

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To sidestep this problem, Merkx and coworkers recently developed a rational strategy for

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construction FRET sensors using self-associating FP variants.31 The group discovered that a S208F

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mutation on both ECFP and EYFP leads to a weak hydrophobic interaction between the FRET pair, and

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a V224L mutation enhances the FRET efficiency (Figure 1e, f). The self-associating ECFP/EYFP pair


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was adapted to the CALWY (CFP-Atox1-Linker-WD4-YFP) sensor for Zn2+, previously reported by the

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same group.32 Although the original sensor had suffered from a small change in emission ratio (dynamic

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range of 21%), the introduction of the S208F/V224L mutations displayed 6-fold improvements in the

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dynamic range. Moreover, this group expanded the self-associating FP strategy to a mOrange/mCherry

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FRET pair.33 The red-shifted “sticky” FRET pair allowed the simultaneous use of spectrally orthogonal

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CALWY sensors to visualize Zn2+ over a broad concentration range in the same cellular compartment.

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This sticky FP strategy has also been employed in FRET sensors to detect protease34, bile acid35, and

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antibodies36. In an analogous manner, Serrano and coworkers reported that the introduction of

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peptide-domain helper module into the intermolecular FRET sensor as a secondary interaction pair

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could enhance its FRET efficiency.37 Given these successful examples, it is likely that the design

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concept based on a subtle interaction between two FP domains can be a generic strategy for constructing

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robust FRET-based biosensors.

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2-2 Single FP-based sensors It is known that the spectral properties of GFP derivatives are affected by the surrounding molecular

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environment of a FP’s chromophore (e.g. pH or local conformational change). This intrinsic feature of

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FPs has been utilized to develop single FP-based biosensor responding to pH38,39, halide anions40 and

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redox.41,42 To target a broader range of analytes, an extrinsic recognition domain can be inserted into

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FPs.43 Upon interacting the analyte, the recognition domain undergoes a conformational change, which

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causes a local conformation change of the FP, resulting in a change in its fluorescent parameters.4,10

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Circularly permutated FPs (cpFPs) facilitates the development of more sensitive single FP sensors.

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cpFPs were constructed by connecting original N and C termini by a short peptide linker and

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regenerating the novel N and C termini at a specific position.44 One representative example of this

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approach is a G-CaMP sensor for Ca2+, which has a M13 peptide and a calmodulin fused to the N- and


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C-termini of a cpEGFP, respectively.45 The G-CaMP has been continuously remodeled to improve

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fluorescent sensitivity and expand the color palette for multicolor imaging of Ca2+ in different organelles,

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such as cytosol, nucleus and mitochondria, of a single cell.46, 47

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A novel strategy to rationally generate single FP sensors has been emerging, which is based on the

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incorporation of unnatural amino acids (UAAs) into a natural chromophore-forming Tyr66 residue of

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FPs through genetic code expansion methods (Figure 2a).48-53 For example, Yun and coworkers

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replaced all tyrosine residues in the GFP with metal-chelating L-DOPA. The GFP-dopa mutant

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functioned as a selective Cu2+ sensor over other metal ions.49 Wang’s group site-specifically

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incorporated a metal binding amino acid, HqAla (2-amino-3-(8-hydroxyquinolin-5-yl)propanoic acid),

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into the Tyr66 of a cpsfGFP variant.50 This construct exhibited a significant (7.2-fold) fluorescence

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increase by Zn2+ in living E coli cells. Chemically reactive-UAAs can also be incorporated to the

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chromophore to generate reaction-based FP sensors. Schultz and coworkers demonstrated a H2O2

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sensitive FP sensor (UFP-Tyr66pBoPhe) by the replacement of Tyr66 of GFP to

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p-borono-L-phenylalanine (pBoPhe) having an arylboronate side chain.51 In the absence of H2O2, this

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sensor doesn’t show fluorescence because the vacant 2p orbital of boron readily accepts electrons to

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make the chromophore more electron-deficient. Upon H2O2 oxidation, pBoPhe is converted to the

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original tyrosine residue, and the fluorescence was recovered. Whereas most reported UAA-based

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biosensors have been limited to use in vitro or in E. coli, it has recently become possible to construct

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them in mammalian cells. Ai’s group developed UAA-based H2S sensor in which Tyr66 was replaced

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with p-azido-L-phenylalanine (pAzF).52 The azide-modified chromophore was selectively reduced by

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H2S, resulting in sensitive fluorescence enhancement. The sensor was successfully expressed in

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mammalian cells by using orthogonal tRNA/aminoacyl-tRNA synthetase pairs, and responded to H2S

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within ~7 min after the addition of 50 µM of NaHS. The Ai group has also developed a UAA-based

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sensor for peroxynitrite, which represents the first genetically encoded peroxynitrite probe that can be

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used in mammalian cells.53 Although UAA-based FP sensors are still in their infancy and have some


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limitations in their reversibility and responsibility, this strategy is expected to provide further structural

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and functional diversity of single FP-based sensors.

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2-3. BiFC sensor In the conventional design of BiFC sensors, a FP is split into two fragments, and fused to

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recognition domains that associate in the presence of an analyte of interest. The two halves of the FP do

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not emit fluorescence in the state of dissociation. Upon the analyte-induced interaction of the

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recognition domains, the complementary fragments of the FP are brought into close proximity and

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reconstitute the β-barrel structure of the FP, resulting in the recovery of the fluorescence signal.54 In

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general, the split FP strategy have a much lower background so that it may give a greater dynamic range

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than those of FRET and single FP sensors. On the other hand, a major drawback in split FPs is its

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irreversibility. While irreversibility provides a significant advantage for detecting transient and/or weak

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interaction, it is unsuitable for analyzing dynamics of a specific analyte. 55,56

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Campbell

and

coworkers

recently

developed

an

alternative

BiFC

sensors

based

on

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dimerization-dependent FPs (ddFPs), which allows reversible fluorescence change by FPs

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complementation (Figure 2b).57 The group focused on the oligomeric property of RFPs that helps

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stabilize the chromophore in a brightly fluorescent state. In the first study, a RFP heterodimer

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(ddRFP-A1B1) derived from Discosoma red FP (DsRed) was generated by library screening strategy.

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The ddRFP exhibited weak fluorescence in the dissociation state, but was 10-fold brighter upon

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heterodimer formation with a Kd of 33 µM. This fluorogenic property of ddRFP was utilized to create

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red intensiometric biosensors, including detection of PPIs, Ca2+ dynamics and protease. This group

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subsequently expanded the color palette of ddFPs by developing ddGFP and ddYFP.58 These constructs

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improved in the brightness and contrast, which allowed imaging of endomembrane proximity between

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endoplasmic reticulum and mitochondria, termed mitochondria-associated membrane. These successful


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examples demonstrated that the ddFP strategy could be promising for novel design format of BiFC

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biosensors.

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2-4. Biosensors based on non-GFP protein scaffolds

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One of the most remarkable advances in recent FP-based biosensors represents the discovery and

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usage of non-GFP types of FPs as a fluorescent transducer, which exhibit characteristic fluorescent

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spectra and emission mechanism distinct from GFP-like proteins. For example, near-infrared (NIR) FPs,

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such as IFP1.419 and iRFP20, have been engineered from bacteriophytochromes, and these proteins

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require biliverdin for chromophore formation.19, 20 Taking advantage of the intrinsic Hg2+ sensitivity of

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IFP1.4, NIR Hg2+ sensor was developed.59 Verkhusha and coworkers exploited a NIR-BiFC reporter

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made of iRFP for detecting PPIs in whole mammals.60 In this work, iRFP was divided into two distinct

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domains, PAS and GAF, and were fused with FRB and FKBP respectively. The rapamycin-induced

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PPIs resulted in a 35-fold fluorescent increase in the HeLa cell. The high penetrability and

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low-background signal of the NIR fluorescence enabled high-contrast visualization of PPIs even in

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living mice. While this iRFP-based BiFC sensor is irreversible, Michnick and coworkers have

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successfully constructed a reversible split BiFC system based on IFP1.4.61 The reversibility was

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demonstrated by spatiotemporal analysis of hormone-induced signaling complexes in living yeast and

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mammalian cells.

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Bacterial and archaeal rhodopsins that have a retinal as a chromophore were engineered for voltage

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sensors.62, 63 This sensing mechanism is dependent on protonation states of the Schiff base, which links

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the retinal to the protein core. A change in membrane potential could alter the local electrochemical

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potential of the proton at the Shiff base, affecting the acid-base equilibrium and inducing rhodopsin’s

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spectral shift. The latest version of the rhodopsin-based voltage sensor is FRET-opsins, in which L.

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maculans rhodopsin is fused with bright GFP-like proteins, such as mCitrine and mOrange2.64 The ACS Paragon Plus Environment

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FRET-opsin sensors enabled the visualizing of neural spiking in brain tissue with high brightness and

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fidelity.

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Another new class of FPs was recently discovered by Miyawaki’s group. They identified and

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isolated a ligand-inducible fluorescent protein from Japanese eel, called UnaG, belonging to the

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fatty-acid-binding protein (FABP) family.24 UnaG exhibited a green fluorescence upon noncovalent,

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strong binding to bilirubin. This feature allowed developing a fluorogenic biosensor for quantifying

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bilirubin from human clinical samples.

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3. Semisynthetic fluorescent biosensors.

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An alternative powerful methodology to create protein-based fluorescent biosensors relies on the

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site-specific chemical modification of a protein framework with synthetic fluorophores. Compared to

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the genetically encoded FP based sensors, such semisynthetic biosensors have three advantages5: 1) the

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smaller fluorophore size should lead to minimum perturbations to the structure and function of the

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protein scaffold, 2) the flexibility to use a wide repertoire of fluorophores with diverse properties, such

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as fluorescence wavelength, brightness, stability against photo-bleaching, microenvironment (e.g. pH,

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solvent polarity) sensitivity, 3) synthetic fluorophores can be incorporated at much more positions in the

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receptor protein.

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Traditionally, semisynthetic biosensors have been constructed in vitro, and introduced into a live

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cell by invasive methods such as microinjection or electroporation, which is likely to cause cell

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damage.65,66 However, with the advent of bioorthogonal reactions and selective protein labeling methods,

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it is now becoming feasible to directly construct semisynthetic biosensors in situ.67-74 In this section, a

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variety of chemical strategies to create semisynthetic biosensors are described.


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3-1. A site-specific modification by genetically incorporated reactive handles

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The nucleophilic thiol of cysteine has often been used as a reactive handle for the site-specific

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modification of proteins with thiol-selective electrophiles, such as maleimides and α-halocarbonyl

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compounds.72,75 The analyte-binding events can be transduced onto a fluorescent change by installing an

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environment-sensitive dye into a protein scaffold at a specific site where conformational or

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microenvironmental changes take place.76,77

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Recently, Rauh and coworkers have established a new HTS system for kinase inhibitors using

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fluorophore-kinase conjugates.9,78 In the earlier study, acrylodan modification of a cysteine in a critical

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regulatory loop region of cSrc kinase enabled development of a direct binding assay for identifying and

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characterizing small-molecule inhibitors that specifically stabilize the inactive form of the kinase. The

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group subsequently improved this system by using red-shift fluorophores to avoid intrinsic inhibitor

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fluorescence that may cause false-positive and –negative results.79 This strategy was extended to

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phosphatases, allowing a HTS assay for ligands of the allosteric pocket of protein tyrosine phosphatase

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1B.80

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Specific PPIs in living cells can be monitored by environment-sensitive fluorophores located within

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or near an interaction interface.81 Recently, Hahn and coworkers have developed a semisynthetic

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biosensor based on a monobody scaffold that can be tailored to bind different targets via HTS assay

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(Figure 3a).82 In this strategy, a library of fibronectin monobodies was screened to find an appropriate

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monobody with the appropriate binding selectivity and affinity for the activated, open form of SH3

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domain of Src-family kinase (SFK). The selected monobody was fused to an environment-sensitive

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fluorophore and an internal standard FP in vitro. Upon binding to the activated SFK, the biosensor

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altered the fluorescence from the environment-sensitive dye. The resulting fluorescence ratio provided a


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quantitative measure of the kinase activity in a live cell microinjected with the sensor. This HTS-based

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method would be powerful for constructing biosensors when no suitable affinity reagents are known.

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Bioorthogonal reactive handles other than mutant Cys can be incorporated into a protein framework

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in the form of UAAs using the expanded genetic code method.83-85 Recently, Chen and coworkers

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introduced an azidocyclopentyl lysine at various sites of the pH-responsive HdeA protein, and labeled

6

with an alkyne-functionalized 4-DMN (4-N,N-dimethylamino-1,8-naphthalimide) by copper-catalyzed

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azide-alkyne cycloaddition (CuAAC), or “click chemistry”, in E. coli and on mammalian cell surface

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(Figure 3b).86 The pH-induced conformational change of HdeA was transduced into an increase in

9

fluorescence. This HdeA-based biosensor represents the first genetically encoded pH indicator that can

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sustain the extremely low pH (pH 7 to 2) that bacterial or mammalian cells might encounter under

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stressful conditions or during pathogenesis.

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3-2. Peptide/Protein-tag based biosensor

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As mentioned above, FRET-based sensors are the most ideal for cellular applications, because they

15

give a ratiometric signal. However, these sensors strongly rely on a conformational change of a protein

16

upon ligand binding, which severely limits the choice of proteins available for the development of new

17

FRET biosensors. Furthermore, since the binding-induced conformational change is usually small, a

18

careful optimization is frequently needed to obtain a satisfactory dynamic range.

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To circumvent such shortcomings, a new class of rationally designed semisynthetic fluorescent sensor,

20

called Snifits (SNAP tag-based indicator proteins with a fluorescent intramolecular tether), has recently

21

been developed by Johnsson and coworkers.87,88 Snifits exploits the SNAP- and CLIP-tag technologies

22

developed by the same group, which can be specifically and covalently labeled with O6-benzylguanine

23

(BG) and O2-benzylcytosine (BC) derivatives, respectively (Figure 4a). Snifits are comprised of 1)

24

SNAP-tag, 2) CLIP-tag (or FPs), 3) an analyte-binding protein. SNAP-tag is specifically modified with ACS Paragon Plus Environment

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a synthetic molecule containing both a fluorophore and an affinity ligand of the analyte-binding protein

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(Figure 4b). CLIP-tag is labeled with a fluorophore that forms a good FRET pair with the fluorophore

3

attached to SNAP-tag. In the absence of analyte, the Snifit is in a closed conformation through the

4

intramolecular ligand binding, whereas in the presence of analyte the equilibrium is shifted towards an

5

open conformation in a competitive binding with the intermolecular ligand (analyte). Such a switch in

6

the relative position of two fluorophores causes a FRET change.

7

As a proof-of-principle, the group employed human carbonic anhydrase (HCA) as the binding protein

8

and benzenesulfonamide as the ligand.87 The sensors, Cy5-SNAP_DY547-CLIP_HCA, ratiometically

9

sensed HCA inhibitors and Zn2+. In the next study, the group demonstrated that the sensing kinetics

10

could be tuned by choosing an appropriate intramolecular ligand.67 The rational optimization of the

11

sensor by the insertion of rigid polyproline linkers allowed an improvement of the sensor’s dynamic

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range from 1.8 to 4.9 in vitro. Furthermore, the optimized sensor was successfully constructed as a

13

fusion to a transmembrane anchor on HEK293T cells, and displayed the maximum ratio change of 3.3

14

upon the ligand binding. More recently, this strategy was expanded to the on-cell sensing of

15

neurotransmitters, including glutamate68, gamma-aminobutyric acid69 and acetylcholine70. These

16

achievements have proven the potential generality of Snifits as a rational design strategy for

17

constructing semisynthetic biosensors.

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3-4. Traceless affinity-based labeling Almost all the existing strategies for the construction of fluorescent biosensors strongly depend on

21

the technologies of genetic manipulation. Although such approaches are undoubtedly powerful, they

22

require the exogenous gene expression or the microinjection of recombinant proteins assembled in vitro,

23

which perturbs the physiological condition of cells. It is no doubt that the direct conversion of


13

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1

endogenous proteins into semisynthetic fluorescent biosensors in their native habitat is one of the most

2

preferable approaches.

3

Toward this end, we focused on an affinity-based protein labeling that offers a highly site-specific

4

and selective protein modification with synthetic probes consisting of a protein ligand and a reactive

5

group. In this approach, the ligand selectively binds to the target protein, driving a chemical reaction of

6

the reactive group with an amino acid located at the vicinity of the ligand-binding pocket through the

7

proximity effect.71,72 A crucial shortcoming, however, is that the labeled product impairs the protein’s

8

function due to the covalent attachment of the ligand, which permanently occupies the active site.89,90

9

A significant breakthrough was achieved by connecting an affinity ligand and a probe through a

10

cleavable phenylsulfonate ester (tosyl) group. In the ligand-directed tosyl (LDT) chemistry, the surface

11

of the target protein can be specifically labeled by an SN2-type reaction with the concurrent release of

12

the ligand molecule, so that the labeled protein retains its native function (Figure 5a).73,91,92 By the

13

combined use of the LDT chemistry with FP-tag technologies, we recently visualized a

14

rapamycin-mediated complexation of endogenous FKBP12 (eFKBP12) and FRB inside of living cells.96

15

Intracellular eFKBP12 was selectively labeled with oregon green (OG) fluorophore by LDT chemistry.

16

The rapamycin-induced interaction of the OG-modified eFKBP12 and FP-tagged FRB in living cells

17

could be monitored through intermolecular FRET signal.

18

We have also developed affinity-guided 4-dimethylaminopyridine (DMAP) catalysts, termed AGD

19

chemistry, that facilitate the specific chemical acylation of proteins with fluorophore-appended

20

thiophenyl ester as an acyl donor.94,95 In the recent study, we demonstrated the selective labeling of

21

bradykinin B2 receptor (B2R), a G-protein coupled receptor, on live cell surfaces.96 The

22

fluorescein-labeled B2R can act as a turn-on fluorescent biosensor for various antagonist candidates

23

using a bimolecular fluorescence quenching and recovery (BFQR) system on the live-cell surfaces

24

(Figure 5b).

25 26

Despite its high target-selectivity and biocompatibility, the LDT chemistry suffered from its slow reaction rate and low labeling efficiency in some situation. The AGD chemistry showed excellent ACS Paragon Plus Environment

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reaction kinetics, but nonspecific labeling derived from the non-catalytic acylation of proteins by the

2

acyl donors caused high-background signals in live-cell imaging. These problems motivated us to

3

develop a ligand-directed acyl imidazole (LDAI) chemistry (Figure 5a).74,97,98 The moderately reactive

4

acyl imidazole allowed the selective and rapid modification of the endogenous folate receptor (eFR) on

5

the surface of live cells, which could not be efficiently labeled using LDT chemistry. More importantly,

6

the LDAI method enabled the direct conversion of the eFR on cell surface into a fluorescent biosensor

7

for sensing its ligands by incorporating fluorescein, which was utilized for the first live cell study of the

8

binding kinetics of FR ligands.74 This example highlighted the utility of the traceless affinity-based

9

labeling as a powerful approach for in situ construction of endogenous protein-based fluorescent

10

biosensors and functional analysis of natural proteins in their native environments.

11 12 13 14

4. Conclusions and outlook We have summarized recent advances in protein-based fluorescent biosensors and their application

15

to live cell imaging. These not only demonstrated the great improvement of existing biosensors, but also

16

provided novel and powerful tools in current biology and pharmacology. One of the important

17

challenges in protein-based fluorescent biosensors is the application of these biosensors in tissues and

18

whole animals. While several FP-based sensors have been used in tissues and transgenic animals, they

19

often suffered from low signal-to-noise ratios owing to its low brightness and/or short wavelength. 1,18

20

The further engineered NIR FPs derived from bacterial phytochromes, more suitable biosensors for deep

21

tissue in vivo imaging, will settle this problem. On the other hand, usage of semisynthetic biosensors in

22

vivo is still limited because of the difficulty in their delivery to whole animals. However, newly

23

emerging strategies, such as Snifits based on the SNAP-tag technology and traceless affinity labeling

24

methods, may have the potential to construct semisynthetic biosensors even in animals as well as in

25

cells.73,88


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1


Another significant challenge is in the simultaneous use of biosensors for multi-parameter imaging

2

in a single cell. Recently, cell-to-cell heterogeneity has been shown to play critical roles for signal

3

transduction.99,100 To address this phenomena, the simultaneous visualizing of multiple analytes in

4

individual cells is essential to more precisely analyze the cellular function in their populations. However,

5

despite the availability of a wide range of fluorescent biosensors for detecting specific biomolecules and

6

events, a limited number of spectrally distinct probes have hampered such multiplexed imaging of

7

individual signaling pathways.21 Therefore, many efforts should be dedicated to further expand the color

8

palette of the fluorescent biosensors and to couple genetic and chemical strategies, which facilitate our

9

understanding of the intricate cellular activities and networks.

10 11 12 13 14 15

KEY WORDS

16

1) Protein-based fluorescent biosensors: integrated devices that convert a molecular-recognition event

17

to a fluorescent signal.

18

2) Live cell imaging: the study of living cells using a fluorescent microscopy.

19

3) Förster resonance energy transfer (FRET): a mechanism describing energy transfer between two

20

fluorophores.

21

4) Fluorescent proteins: protein family that shares the unique property of emitting a visible

22

fluorescence.

23

5) Fluorescent dyes: fluorescent chemical compounds.

24

6) Unnatural amino acids: non-naturally encoded amino acids that are chemically synthesized.


16


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1

7) SNAP-tag: a self-labeling protein tag (19.4 kDa) commercially available in various expression

2

vectors. SNAP-tag can be fused to any protein of interest, covalently tagging that protein with

3

O6-benzylguanine derivatives.

4

8) Affinity-based labeling: a method to chemically modify an amino acid residue within a specific

5

ligand-binding site of a protein using a labeling reagent consisting of a protein ligand and a reactive

6

group.

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AUTHOR INFORMATION

9

Corresponding Author

10

Email: [email protected]

11

Note

12

The authors declare no competing financial interest.

13 14

ACKNOWLEDGEMENTS

15

T.T. thanks J.-L. Chaubard for helpful discussions and advice on the manuscript. T.T. also acknowledge

16

the Japan Society for the Promotion of Science (JSPS) Fellowships for Young Scientists. This work was

17

funded by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and

18

Technology (CREST) to I.H.

19 20


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1

2 3 4 5 6 7 8 9 10


FIGURES

Figure 1. Representative design formats for FRET-based biosensor. (a) A single binding domain undergoes a conformational change upon binding analyte. (b) Biosensors to monitor post-translational modifications. (c) Biosensors to detect protease activity. (d) Biosensors based on an analyte-dependent protein-protein interaction. (e, f) FRET-based sensors based on self-associating FPs. (e) Protease sensor. (f) eCALWY sensor for Zn2+.


18


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Page 20 of 31

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2 3 4 5 6

Figure 2. (a) Single FP biosensors containing unnatural amino acids (UAAs) in their chromophore. (b) Dimerization-dependent fluorescent proteins (ddFPs)-based biosensor.


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1 2 3 4 5


Figure 3. (a) A semisynthetic biosensor for Src-family kinase (SFK) activity. (b) HdeA-based semisynthetic biosensors for monitoring a wide range of pH changes (pH 7 ~ 2).


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Page 22 of 31

Figure 4. (a) Labeling mechanism of SNAP-tag. (b) Schematic illustration of the principle of Snifits sensor.


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1 2 3 4 5 6 7


Figure 5. Conversion of native (endogenous) proteins into semisynthetic fluorescent biosensors in cells by (a) LDT, LDAI, and (b) AGD chemistry.


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Recent Progress in Lectin-Based Biosensors.

Yeast-based biosensors: design and applications.

Recent Progress in Fluorescent Imaging Probes.

Recent Progress of Microfluidics in Translational Applications.

Biosensors and their applications - A review.

Recent progress in design, synthesis, and applications of one-dimensional TiO2 nanostructured surface heterostructures: a review.

Ratiometric mechanosensitive fluorescent dyes: Design and applications.

Influenza: recent advancements and their clinical applications.

Recent progress in metal-organic complexes for optoelectronic applications.

Recent Progress in Ferrocene-Modified Thin Films and Nanoparticles for Biosensors.

Progress of Mimetic Enzymes and Their Applications in Chemical Sensors.

Flexible energy-storage devices: design consideration and recent progress.

Editorial: fluorescent biosensors.

An Overview on Recent Progress in Electrochemical Biosensors for Antimicrobial Drug Residues in Animal-Derived Food.

Recent Advances in Silicon Nanowire Biosensors: Synthesis Methods, Properties, and Applications.

Protein-directed synthesis of pH-responsive red fluorescent copper nanoclusters and their applications in cellular imaging and catalysis.

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications.

Recent development in partial differential equations and their applications.

Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging.

Selective homocysteine turn-on fluorescent probes and their bioimaging applications.

Recent trends of biosensors in Japan.

neural network: design, analysis, and applications.

Development and Applications of Portable Biosensors.

Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors.