Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

A fast, high-affinity fluorescent serotonin biosensor engineered from a tick lipocalin.

Serotonin (5-HT) is an important signaling monoamine and neurotransmitter. We report structure-guided engineering of a green fluorescent, genetically encoded serotonin sensor (G-GESS) from a 5-HT-binding lipocalin in the soft tick Argas monolakensis. G-GESS shows fast response kinetics and high affinity, specificity, brightness and photostability. We used G-GESS to image 5-HT dynamics in cultured cells, brain slices and behaving mice.

How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger.

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

Structural origin and rational development of bright red noncanonical variants of green fluorescent protein.

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent proteins (RFPs) have been rare. The 3-aminotyrosine modified superfolder green fluorescent protein (aY-sfGFP) represents a recent advance, yet the molecular mechanism for its red-shifted fluorescence remains elusive while its dim fluorescence hinders applications. Herein, we implement femtosecond stimulated Raman spectroscopy to obtain structural fingerprints in the electronic ground state and reveal that aY-sfGFP possesses a GFP-like instead of RFP-like chromophore. Red color of aY-sfGFP intrinsically arises from a unique "double-donor" chromophore structure that raises ground-state energy and enhances charge transfer, notably differing from the conventional conjugation mechanism. We further developed two aY-sfGFP mutants (E222H and T203H) with significantly improved (∼12-fold higher) brightness by rationally restraining the chromophore's nonradiative decay through electronic and steric effects, aided by solvatochromic and fluorogenic studies of the model chromophore in solution. This study thus provides functional mechanisms and generalizable insights into ncAA-RFPs with an efficient route for engineering redder and brighter fluorescent proteins.

Improved Red Fluorescent Redox Indicators for Monitoring Cytosolic and Mitochondrial Thioredoxin Redox Dynamics.

Thioredoxin (Trx) is one of the major thiol-dependent antioxidants in living systems. The study of Trx functions in redox biology was impeded by the lack of practical tools to track Trx redox dynamics in live cells. Our previous work developed TrxRFP1, the first genetically encoded fluorescent indicator for Trx redox. In this work, we report an improved fluorescent indicator, TrxRFP2, for tracking the redox of Trx1, which is primarily cytosolic and nuclear. Furthermore, because mitochondria specifically express Trx2, we have created a new genetically encoded fluorescent indicator, MtrxRFP2, for the redox of mitochondrial Trx. We characterized MtrxRFP2 as a purified protein and used subcellularly localized MtrxRFP2 to image mitochondrial redox changes in live cells.

Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo.

The NanoLuc luciferase (NLuc) and its furimazine (FRZ) substrate have revolutionized bioluminescence (BL) assays and imaging. However, the use of the NLuc-FRZ luciferase-luciferin pair for mammalian tissue imaging is hindered by the low tissue penetration of the emitting blue photons. Here, we present the development of an NLuc mutant, QLuc, which catalyzes the oxidation of a synthetic QTZ luciferin for bright and red-shifted emission peaking at ∼585 nm. Compared to other small single-domain NLuc mutants, this amber-light-emitting luciferase exhibited improved performance for imaging deep-tissue targets in live mice. Leveraging this novel bioluminescent reporter, we further pursued in vivo immunobioluminescence imaging (immunoBLI), which used a fusion protein of a single-chain variable antibody fragment (scFv) and QLuc for molecular imaging of tumor-associated antigens in a xenograft mouse model. As one of the most red-shifted NLuc variants, we expect QLuc to find broad applications in noninvasive mammalian imaging. Moreover, the immunoBLI method complements immunofluorescence imaging and immuno-positron emission tomography (immunoPET), serving as a convenient and nonradioactive molecular imaging tool for animal models in basic and preclinical research.

A general strategy to red-shift green fluorescent protein-based biosensors.

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic ß-cells.

Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity.

Redox-active molecules play essential roles in cell homeostasis, signaling, and other biological processes. Dysregulation of redox signaling can lead to toxic effects and subsequently cause diseases. Therefore, real-time tracking of specific redox-signaling molecules in live cells would be critical for deciphering their functional roles in pathophysiology. Fluorescent protein (FP)-based genetically encoded redox indicators (GERIs) have emerged as valuable tools for monitoring the redox states of various redox-active molecules from subcellular compartments to live organisms. In the first section of this review, we overview the background, focusing on the sensing mechanisms of various GERIs. Next, we review a list of selected GERIs according to their analytical targets and discuss their key biophysical and biochemical properties. In the third section, we provide several examples which applied GERIs to understanding redox signaling and oxidative toxicology in pathophysiological processes. Lastly, a summary and outlook section is included.

Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging.

Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular showed superior in vitro and in vivo sensitivity over commonly used bioluminescence reporters. We adapted this pair to develop a bioluminescence resonance-energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

Identification of Factors Complicating Bioluminescence Imaging.

In vivo bioluminescence imaging (BLI) has become a standard, non-invasive imaging modality for following gene expression or the fate of proteins and cells in living animals. Currently, bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Inconsistent results of experiments using different bioluminescent reporters, such as Akaluc and Antareas2, have been reported. Herein, we re-examined the inconsistency in several experimental settings and identified the factors, such as ATP dependency, stability in serum, and molecular sizes of luciferases, that contributed to the observed differences. We expect this study will make the research community aware of these factors and facilitate more accurate interpretation of BLI data by considering the nature of each bioluminescent reporter.

Genetically Encoded, Photostable Indicators to Image Dynamic Zn2+ Secretion of Pancreatic Islets.

As an essential element for living organisms, zinc (Zn2+) exerts its biological functions both intracellularly and extracellularly. Previous studies have reported a number of genetically encoded Zn2+ indicators (GEZIs), which have been widely used to monitor Zn2+ in the cytosol and intracellular organelles. However, it is challenging to localize existing GEZIs to the extracellular space to detect secreted Zn2+. Herein, we report two photostable, green fluorescent protein (GFP) based indicators, ZIBG1 and ZIBG2, which respond to Zn2+ selectively and have affinities suited for detecting Zn2+ secretion from intracellular vesicles. In particular, ZIBG2 can be effectively targeted to the extracellular side of plasma membrane. We applied cell surface-localized ZIBG2 to monitor glucose-induced dynamic Zn2+ secretion from mouse insulinoma MIN6 cells and primary mouse and human pancreatic islets. Because Zn2+ is co-released with insulin from ß-cells, the fluorescence of cell surface-localized ZIBG2 was shown to be a strong indicator for the functional potency of islets. Our work here has thus expanded the use of GEZIs to image dynamic Zn2+ secretion in live tissue. Because it is convenient to use genetically encoded indicators for expression over extended periods and for in vivo delivery, we envision future applications of ZIBG2 in development of induced ß-cells or islets to advance cell replacement therapies for diabetes and in direct imaging of Zn2+ secretion dynamics in vivo.

Molecular Tools to Generate Reactive Oxygen Species in Biological Systems.

Reactive oxygen species (ROS) not only are byproducts of aerobic respiration, but also play vital roles in metabolism regulation and signal transductions. It is important to understand the functions of ROS in biological systems. In addition, scientists have made use of ROS to kill bacteria and tumors through a process known as photodynamic therapy (PDT). This paper provides a concise review of current molecular tools that can generate ROS in biological systems via either nongenetic or genetically encoded way. Challenges and perspectives are further discussed with the hope of broadening the applications of ROS generators in research and clinical settings.

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

The N-B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite.

Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

Single Fluorescent Protein-Based Indicators for Zinc Ion (Zn(2+)).

Genetically encoded fluorescent Zn(2+) indicators (GEZIs) are highly attractive research tools for studying Zn(2+) homeostasis and signaling in mammalian cells. Most current GEZIs are based on Förster resonance energy transfer (FRET) between a select pair of fluorescent proteins (FPs) fused with Zn(2+)-binding motifs. One drawback of such FRET-based GEZIs is their broad spectral profile bandwidths, creating challenges when monitoring multiple targets or parameters. To address this issue, we have engineered a group of intensiometric GEZIs based on single teal and red FPs that can be utilized to monitor subcellular Zn(2+) diffusion and glucose-induced Zn(2+) secretion in pancreatic INS-1E ß-cells. These GEZIs offer the simplicity of intensiometric measurements, compatibility in multicolor imaging, large dynamic ranges, and relatively small molecular sizes, making them valuable additions to the molecular toolbox for imaging Zn(2+).

A membrane-activatable near-infrared fluorescent probe with ultra-photostability for mitochondrial membrane potentials.

Mitochondrial membrane potential (MMP) is a frequently used indicator for mitochondrial function. Herein, we report a photostable near-infrared (NIR) fluorescent dye for monitoring MMP. This new probe, named NIMAP, is non-fluorescent in aqueous solution and can be activated by cell membranes, providing high fluorescence contrast and low background fluorescence. NIMAP has been validated for monitoring MMP in living mammalian cells and in mice. Due to the large fluorescence response, low fluorescence background, high photostability, and excellent tissue penetration resulting from red-shifted excitation and emission in the "optical window" above 600 nm, broad applications of this new probe are expected.

Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells.

Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.

Light activation of protein splicing with a photocaged fast intein.

Intein-mediated protein splicing has found broad biotechnological applications. Herein, we describe our recent result in engineering a photoactivatable intein compatible with living mammalian cells. A photocaged cysteine amino acid residue was genetically introduced into a highly efficient Nostoc punctiforme (Npu) DnaE intein. The resulting photocaged intein was inserted into a red fluorescent protein (RFP) mCherry and a human Src tyrosine kinase to create inactive chimeric proteins. A light-induced photochemical reaction was able to reactivate the intein and trigger protein splicing. Active mCherry and Src were formed as observed by direct fluorescence imaging or imaging of an Src kinase sensor in mammalian cells. The genetically encoded photocaged intein is a general optogenetic tool, allowing effective photocontrol of primary structures and functions of proteins.