ATP-Independent Water-Soluble Luciferins Enable Non-Invasive High-Speed Video-Rate Bioluminescence Imaging of Mice.

NanoLuc luciferase and its derivatives are attractive bioluminescent reporters recognized for their efficient photon production and ATP independence. However, utilizing them for in vivo imaging poses notable challenges. Low substrate solubility has been a prominent problem, limiting in vivo brightness, while substrate instability hampers consistent results and handling. To address these issues, we developed a range of caged PEGylated luciferins with improved stability and water solubility of up to 25 mM, resulting in substantial bioluminescence increases in mouse models. This advancement has created the brightest and most sensitive luciferase-luciferin combination, enabling high-speed video-rate imaging of freely moving mice with brain-expressed luciferase. Furthermore, we developed a bioluminescent Ca 2+ indicator with exceptional sensitivity to physiological Ca 2+ changes and paired it with a new substrate to showcase non-invasive, video-rate imaging of Ca 2+ activity in a defined brain region in awake mice. These innovative substrates and the Ca 2+ indicator are poised to become invaluable resources for biological and biomedical fields.

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.

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.

Engineering and Characterization of 3-Aminotyrosine-Derived Red Fluorescent Variants of Circularly Permutated Green Fluorescent Protein.

Introducing 3-aminotyrosine (aY), a noncanonical amino acid (ncAA), into green fluorescent protein (GFP)-like chromophores shows promise for achieving red-shifted fluorescence. However, inconsistent results, including undesired green fluorescent species, hinder the effectiveness of this approach. In this study, we optimized expression conditions for an aY-derived cpGFP (aY-cpGFP). Key factors like rich culture media and oxygen restriction pre- and post-induction enabled high-yield, high-purity production of the red-shifted protein. We also engineered two variants of aY-cpGFP with enhanced brightness by mutating a few amino acid residues surrounding the chromophore. We further investigated the sensitivity of the aY-derived protein to metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS). Incorporating aY into cpGFP had minimal impact on metal ion reactivity but increased the response to RNS. Expanding on these findings, we examined aY-cpGFP expression in mammalian cells and found that reductants in the culture media significantly increased the red-emitting product. Our study indicates that optimizing expression conditions to promote a reduced cellular state proved effective in producing the desired red-emitting product in both E. coli and mammalian cells, while targeted mutagenesis-based protein engineering can further enhance brightness and increase method robustness.

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

Development, Characterization, and Structural Analysis of a Genetically Encoded Red Fluorescent Peroxynitrite Biosensor.

Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p-boronophenylalanine (pBoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced pBoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitro and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11B-NMR, and computational methods. The boron atom in pnRFP adopts an sp2-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed "turn-off" fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.

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.

A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn2.

Synaptic zinc ion (Zn2+) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn2+ in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn2+. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn2+ dynamics in the nervous system. Our engineered far-red fluorescent indicator for synaptic Zn2+ (FRISZ) displayed a substantial Zn2+-specific turn-on response and low-micromolar affinity. We genetically anchored FRISZ to the mammalian extracellular membrane via a transmembrane (TM) ⍺ helix and characterized the resultant FRISZ-TM construct at the mammalian cell surface. We used FRISZ-TM to image synaptic Zn2+ in the auditory cortex in acute brain slices and awake mice in response to electric and sound stimuli, respectively. Thus, this study establishes a technology for studying the roles of synaptic Zn2+ in the nervous system.

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

There is great interest in developing boronolectins, which are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin, which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid-and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

Illuminating lactate in cells, mice, and patient samples.

Lactate has emerged as a central metabolic fuel and an important signaling molecule. In this issue of Cell Metabolism, Li et al. develop a high-quality lactate sensor, allowing them to monitor lactate levels in cells, subcellular organelles, live mice, and human body fluids.

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.

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 luciferase prosubstrate and a red bioluminescent calcium indicator for imaging neuronal activity in mice.

Although fluorescent indicators have been broadly utilized for monitoring bioactivities, fluorescence imaging, when applied to mammals, is limited to superficial targets or requires invasive surgical procedures. Thus, there is emerging interest in developing bioluminescent indicators for noninvasive mammalian imaging. Bioluminescence imaging (BLI) of neuronal activity is highly desired but hindered by insufficient photons needed to digitalize fast brain activities. In this work, we develop a luciferase prosubstrate deliverable at an increased dose and activated in vivo by nonspecific esterase. We further engineer a bright, bioluminescent indicator with robust responsiveness to calcium ions (Ca2+) and appreciable emission above 600 nm. Integration of these advantageous components enables the imaging of the activity of neuronal ensembles in awake mice minimally invasively with excellent signal-to-background and subsecond temporal resolution. This study thus establishes a paradigm for studying brain function in health and disease.

Engineering and exploiting synthetic allostery of NanoLuc luciferase.

Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein's active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini.

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.

Ratiometric Imaging of Mitochondrial Hydrogen Peroxide in Aß42-Mediated Neurotoxicity.

Oxidative stress is important for the etiology and pathogenesis of Alzheimer's disease (AD). Research tools that can conveniently evaluate oxidative stress in AD models are expected to catalyze and accelerate research on AD. This study explored the use of genetically encoded fluorescent indicators (GEFIs) to detect mitochondrial oxidative stress in organotypic brain slices and AD mouse models. To enable ratiometric normalization and avoid tissue autofluorescence, we genetically fused a green fluorescent hydrogen peroxide (H2O2) indicator, HyPer7, with each of two selected, bright red fluorescent proteins (RFPs), mScarlet-I and tdTomato. The resultant indicators, namely, HyPerGRS and HyPerGRT, were tagged with mitochondrial targeting sequences and examined for localization and function in cultured HeLa cells and primary mouse neurons. We further utilized HyPerGRT, which is a genetic fusion of HyPer7 with tdTomato, to monitor mitochondrial H2O2 in response to the human ß-amyloid 1-42 isoform (Aß42) in cultured brain slices and an AD mouse model. Owing to the high sensitivity and low autofluorescence interference resulting from HyPerGRT, we successfully detected Aß42-mediated mitochondrial H2O2 in these AD models. The results suggest that HyPerGRT is a valuable tool for studying mitochondrial oxidative stress in tissues and animals.

Circularly Permuted Far-Red Fluorescent Proteins.

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the "optical window" above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

Genetically Encoded Green Fluorescent Biosensors for Monitoring UDP-GlcNAc in Live Cells.

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar used by glycosyltransferases to synthesize glycoproteins, glycosaminoglycans, glycolipids, and glycoRNA. UDP-GlcNAc also serves as the donor substrate for forming O-GlcNAc, a dynamic intracellular protein modification involved in diverse signaling and disease processes. UDP-GlcNAc is thus a central metabolite connecting nutrition, metabolism, signaling, and disease. There is a great interest in monitoring UDP-GlcNAc in biological systems. Here, we present the first genetically encoded, green fluorescent UDP-GlcNAc sensor (UGAcS), an optimized insertion of a circularly permuted green fluorescent protein (cpGFP) into an inactive mutant of an Escherichia coli UDP-GlcNAc transferase, for ratiometric monitoring of UDP-GlcNAc dynamics in live mammalian cells. Although UGAcS responds to UDP-GlcNAc quite selectively among various nucleotide sugars, UDP and uridine triphosphate (UTP) interfere with the response. We thus developed another biosensor named UXPS, which is responsive to UDP and UTP but not UDP-GlcNAc. We demonstrated the use of the biosensors to follow UDP-GlcNAc levels in cultured mammalian cells perturbed with nutritional changes, pharmacological inhibition, and knockdown or overexpression of key enzymes in the UDP-GlcNAc synthesis pathway. We further utilized the biosensors to monitor UDP-GlcNAc concentrations in pancreatic MIN6 ß-cells under various culture conditions.

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.