Activity-Based Genetically Encoded Fluorescent and Luminescent Probes for Detecting Formaldehyde in Living Cells

Formaldehyde (FA) is endogenously produced in living systems through a variety of biological processes and has been implicated in many pathological conditions. Detection tools for biological FA are therefore of great interest. Reported here are novel activity-based genetically encoded fluorescent and luminescent probes for detecting FA in aqueous solutions and living mammalian cells. A FA-reactive lysine analogue, PrAK, was site-specifically incorporated into the essential lysine sites of enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc) to afford fluorescent and luminescent FA probes, respectively. FA selectively reacts with PrAK residues on EGFP and fLuc through a 2-aza-Cope rearrangement, resulting in fluorescence and luminescence turn-on responses, respectively, to FA selectively over potentially interfering reactive species in aqueous buffer. Moreover, the genetically encoded probes are capable of visualizing FA at physiologically relevant levels in living mammalian cells by fluorescence and luminescence imaging, demonstrating their potential as new tools to explore FA biology.     

Reaction-based genetically encoded fluorescent hydrogen sulfide sensors

The detection of hydrogen sulfide (H(2)S), a toxic gas and an important biological signaling molecule, has been a long-time challenge. Here we report genetically encoded fluorescent protein (FP)-based probes that can selectively detect H(2)S. By expanding the genetic codes of E. coli and mammalian cells, FP chromophores were modified with the sulfide-reactive azide functional group. These structurally modified chromophores were selectively reduced by H(2)S, resulting in sensitive fluorescence enhancement detectable by spectroscopic and microscopic techniques. Exploration of a circularly permuted FP led to an improved sensor with faster responses, and the feasibility of using such a genetically encoded probe to monitor H(2)S in living mammalian cells has also been demonstrated.     

The Remarkable Effect of Halogen Substitution on the Membrane Transport of Fluorescent Molecules in Living Cells

Small‐molecule‐based fluorescent probes have become important tools in biology for sensing and imaging applications. However, the biological applications of many of the fluorescent molecules are hampered by low cellular uptake and high toxicity. In this paper, we show for the first time that the introduction of halogen atoms enhances the cellular uptake of fluorescent molecules and the nature of halogen atoms plays a crucial role in the plasma membrane transport in mammalian cells. The remarkably higher uptake of iodinated compounds compared to that of their chloro or bromo analogues suggests that the strong halogen bonding ability of iodine atoms may play an important role in the membrane transport. This study provides a novel strategy for the transport of fluorescent molecules across the plasma membrane in living cells.     

Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo

Increasing evidence has highlighted the endogenous production of formaldehyde (FA) in a variety of fundamental biological processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiological and pathological relevance and functions of FA, fluorescent probes for FA imaging in live biological samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theoretical calculations were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-determining step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative β-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aqueous solutions and live cells. Among these probes, the near-infrared probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chemical tools for fluorescence imaging and biological investigation of FA in living systems.

Systematic investigation of various homoallylamines reveals N-p-methoxybenzyl homoallylamine as the optimal 2-aza-Cope reaction moiety for development of highly efficient formaldehyde fluorescent probes for in vitro and in vivo imaging.     

Use of Lanthanide‐Containing Polyoxometalates to Sensitise the Emission of Fluorescent Labelled Serum Albumin

Monitoring the interaction of biomolecules is important, and the use of energy transfer is a principal technique in elucidating nanoscale interactions. Lanthanide compounds are promising luminescent probes for biological samples as their emission is longer‐lived than any native autofluorescence. Polyoxometalates (POMs) are interesting structural motifs to incorporate lanthanides, offering low toxicity and a size pertinent for biological applications. Here, we employ iso‐structured POMs containing either terbium or europium and assess their interaction with serum albumin by sensitisation of a fluorescent tag on the protein via LRET (luminescence resonance energy transfer) by exciting the lanthanide. Time‐resolved measurements showed energy transfer with an efficiency of over 90 % for the POM–protein systems. The Tb–POM results were relatively straightforward, while those with the iso‐structured Eu–POM were complicated by the effect of protein shielding from the aqueous environment.     

Detecting mitochondrial RNA and other cellular events in living cells

Intracellular signaling can be monitored in vivo in living cells by genetically encoded intracellular fluorescent probes. In this review, three aspects of these probes are introduced: 1) the imaging dynamics of endogenous mitochondrial RNA; 2) nuclear receptor and coactivator/corepressor interactions, and; 3) the signal sequence in mitochondrial intermembrane space. These probes are generally applicable to fundamental biological studies as well as for assaying and screening possible pharmaceutical or toxic chemicals that facilitate or inhibit cellular signaling pathways.     

Development of a novel terbium(III) chelate-based luminescent probe for highly sensitive time-resolved luminescence detection of hydroxyl radical

Time-resolved (or time-gated) luminescence detection technique using lanthanide chelates as luminescent probes is a widely used and highly sensitive method for the biological applications. The developments of various functional lanthanide probes that can selectively recognize the biological targets are the essential objective of the technique. In this work, a unique Tb³⁺ chelate-based luminescent probe, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-(p-aminophenoxy)methylene-pyridine] tetrakis(acetate)-Tb³⁺(BMPTA-Tb³⁺), has been designed and synthesized for highly selective and sensitive time-resolved luminescence detection of one highly reactive oxygen species (ROS), hydroxyl radical (OH). The probe is almost non-luminescent, and can selectively react with hydroxyl radical to afford a highly luminescent Tb³⁺ chelate, N,N,N¹,N¹-[2,6-bis(3′-aminomethyl-1′-pyrazolyl)-4-hydroxymethyl-pyridine] tetrakis(acetate)-Tb³⁺ (BHTA-Tb³⁺), accompanied by a 49-fold increase in luminescence quantum yield with a long luminescence lifetime (2.76 ms). The luminescence response of the probe to hydroxyl radical is highly selective and insensitive to pH in the physiological pH range. For loading the probe into the living cells, the acetoxymethyl ester of BMPTA-Tb³⁺ was synthesized and used for the HeLa cell loading. Based on this probe, a background-free time-resolved luminescence imaging method for detecting hydroxyl radical in living cells was successfully established.     

Semisynthesis of fluorescent metabolite sensors on cell surfaces

Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.     

Poly(arginine)‐Selective Coprecipitation Properties of Self‐Assembling Apoferritin and Its Tb3+ Complex: A New Luminescent Biotool for Sensing of Poly(arginine) and Its Protein Conjugates

The apoferritin protein and apoferritin–Tb³⁺ complex were demonstrated to form oligomeric and polymeric self‐assemblies in neutral aqueous solutions, based on characterization by using luminescence and UV/Vis spectroscopy, dynamic light scattering, and transmission electron microscopy. Addition of a 20‐mer or higher poly(arginine) to the solution resulted in coprecipitation through nanoscale interactions, while biological proteins and other poly(amino acids) rarely yielded precipitates under the conditions employed. The apoferritin–Tb³⁺ complex assembly exhibited a particularly long‐lived green luminescence in aqueous solution, and its poly(arginine)‐selective precipitation behavior was followed by monitoring the changes in luminescence. The poly(arginine)‐tagged albumin precipitated selectively and quantitatively, so that the apoferritin–Tb³⁺ complex can function as a new luminescent biotool for the sensing of poly(arginine) and its protein conjugates.     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

New rhodamine nitroxide based fluorescent probes for intracellular hydroxyl radical identification in living cells

The synthesis, characteristics, and biological applications of a series of new rhodamine nitroxide fluorescent probes that enable imaging of hydroxyl radicals (?OH) in living cells are described. These probes are highly selective for ?OH in aqueous solution, avoiding interference from other reactive oxygen species (ROS), and they facilitate ?OH imaging in biologically active samples. The robust nature of these probes (high specificity and selectivity, and facile synthesis) offer distinct advantages over previous methods for ?OH detection.     

o‐Fluorination of Aromatic Azides Yields Improved Azido‐Based Fluorescent Probes for Hydrogen Sulfide: Synthesis,Spectra, and Bioimaging

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple biological functions. To visualize the endogenous in situ production of H₂S in real time, new coumarin‐ and boron‐dipyrromethene‐based fluorescent turn‐on probes were developed for fast sensing of H₂S in aqueous buffer and in living cells. Introduction of a fluoro group in the ortho position of the aromatic azide can lead to a greater than twofold increase in the rate of reaction with H₂S. On the basis of o‐fluorinated aromatic azides, fluorescent probes with high sensitivity and selectivity toward H₂S over other biologically relevant species were designed and synthesized. The probes can be used to in situ to visualize exogenous H₂S and D ‐cysteine‐dependent endogenously produced H₂S in living cells, which makes them promising tools for potential applications in H₂S biology.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).     

聚甲基丙烯酰胺包覆的ZnO发光量子点及其在细胞成像中的应用

通过溶胶-凝胶法合成了在水溶液中稳定发光、荧光颜色可调的ZnO@polymer核壳型纳米粒子, 并研究了这种量子点对人类宫颈癌HeLa细胞的毒性以及细胞吞噬后激光共聚焦成像的效果. 这种荧光探针的外壳是一种通过配位键与ZnO内核结合的共聚物, 该共聚物外层是亲水的聚甲基丙烯酰胺, 内层是疏水的聚甲基丙烯酸酯. 细胞毒性实验证明, 该材料有良好的生物兼容性, 适用于生物荧光标记. 共聚焦荧光成像结果显示, 这些纳米粒子可以穿透HeLa细胞膜并在细胞质中稳定发光.     

Blue-emissive upconversion nanoparticles for low-power-excited bioimaging in vivo

Water-soluble upconversion luminescent (UCL) nanoparticles based on triplet-triplet annihilation (TTA) were successfully prepared by coloading sensitizer (octaethylporphyrin Pd complex) and annihilator (9,10-diphenylanthracene) into silica nanoparticles. The upconversion luminescence quantum yield of the nanoparticles can be as high as 4.5% in aqueous solution. As determined by continuous kinetic scan, the nanoparticles have excellent photostability. Such TTA-based upconversion nanoparticles show low cytotoxicity and were successfully used to label living cells with very high signal-to-noise ratio. UCL imaging with the nanoparticles as probe is capable of completely eliminating background fluorescence from either endogenous fluorophores of biological sample or the colabeled fluorescent probe. In particular, such blue-emissive upconversion nanoparticles were successfully applied in lymph node imaging in vivo of living mouse with excellent signal-to-noise ratio (>25), upon low-power density excitation of continuous-wave 532 laser (8.5 mW cm(-2)). Such high-contrast and low-power excited bioimaging in vivo with a blue-emissive upconversion nanoparticle as probe may extend the arsenal of currently available luminescent bioimaging in vitro and in vivo.     

A Vinyl Sulfone‐Based Fluorogenic Probe Capable of Selective Labeling of PHGDH in Live Mammalian Cells

Chemical probes are powerful tools for interrogating small molecule‐target interactions. With additional fluorescence Turn‐ON functionality, such probes might enable direct measurements of target engagement in live mammalian cells. DNS‐pE (and its terminal alkyne‐containing version DNS‐pE2) is the first small molecule that can selectively label endogenous 3‐phosphoglycerate dehydrogenase (PHGDH) from various mammalian cells. Endowed with an electrophilic vinyl sulfone moiety that possesses fluorescence‐quenching properties, DNS‐pE/DNS‐pE2 became highly fluorescent only upon irreversible covalent modification of PHGDH. With an inhibitory property (in vitro K_i=7.4 μm ) comparable to that of known PHGDH inhibitors, our probes thus offer a promising approach to simultaneously image endogenous PHGDH activities and study its target engagement in live‐cell settings.     

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small‐molecule inhibitors of DOT1L such as FED1 are potential anti‐cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo‐reactive and “clickable” affinity‐based probes (AfBPs), P1 and P2 , which were cell‐permeable and structural mimics of FED1 . The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub‐cellular localization properties of the probes were subsequently studied in live‐cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1 . Finally with P1 / P2 , large‐scale cell‐based proteome profiling, followed by quantitative LC‐MS/MS, was carried out to identify potential cellular off‐targets of FED1 . Amongst the more than 100 candidate off‐targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off‐target of FED1 by preliminary validation experiments including pull‐down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).     

Recent advances in mitochondria-and lysosomes-targeted small-molecule two-photon fluorescent probes

This review summarized the recent advances in small-molecule two-photon fl uorescent probes for monitoring a wide variety of biomolecules and changes inside micro-environment in mitochondria and lysosomes, or served as mitotracker and lysotracker with the assistance of two-photon microscopy.     

Genetically encoded fluorescent reporters of histone methylation in living cells

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.     

Fluorescent Detection of Hypochlorous Acid from Turn‐On to FRET‐Based Ratiometry by a HOCl‐Mediated Cyclization Reaction

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation‐associated diseases. Therefore, the detection of HOCl is of great importance. In this work, we describe the HOCl‐promoted cyclization of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles, which is then exploited as a novel design strategy for the development of a new fluorescence turn‐on HOCl probe 2 . On the basis of the fluorescence resonance energy transfer (FRET) signaling mechanism, 2 was further converted into 1 a and 1 b , which represent the first paradigm of FRET‐based ratiometric fluorescent HOCl probes. The outstanding features of 1 a and 1 b include well‐resolved emission peaks, high sensitivity, high selectivity, good functionality at physiological pH, rapid response, low cytotoxicity, and good cell‐membrane permeability. Furthermore, these excellent attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced HOCl in living cells by using these novel ratiometric probes. We expect that 1 a and 1 b will be useful molecular tools for studies of HOCl biology. In addition, the HOCl‐promoted cyclization reaction of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles should be widely applicable for the development of different types of fluorescent HOCl probes.