From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules

The explosion in genome‐wide sequencing has revealed that noncoding RNAs are ubiquitous and highly conserved in biology. New molecular tools are needed for their study in live cells. Fluorescent RNA–small molecule complexes have emerged as powerful counterparts to fluorescent proteins, which are well established, universal tools in the study of proteins in cell biology. No naturally fluorescent RNAs are known; all current fluorescent RNA tags are in vitro evolved or engineered molecules that bind a conditionally fluorescent small molecule and turn on its fluorescence by up to 5000‐fold. Structural analyses of several such fluorescence turn‐on aptamers show that these compact (30–100 nucleotides) RNAs have diverse molecular architectures that can restrain their photoexcited fluorophores in their maximally fluorescent states, typically by stacking between planar nucleotide arrangements, such as G‐quadruplexes, base triples, or base pairs. The diversity of fluorogenic RNAs as well as fluorophores that are cell permeable and bind weakly to endogenous cellular macromolecules has already produced RNA–fluorophore complexes that span the visual spectrum and are useful for tagging and visualizing RNAs in cells. Because the ligand binding sites of fluorogenic RNAs are not constrained by the need to autocatalytically generate fluorophores as are fluorescent proteins, they may offer more flexibility in molecular engineering to generate photophysical properties that are tailored to experimental needs.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

Fluorogenic ‘click-on’ dendrimer reporter for rapid profiling of cell proliferation

The application of small molecule fluorescent reporters to monitor biological systems is limited by their poor water solubility and background fluorescence of these reporters. Herein, we describe the synthesis and testing of a fluorogenic ‘click’ dendrimer reporter to monitor cellular processes. The reporter system consists of a polyamidoamine (PAMAM) dendrimer conjugated with 3-azido-7-hydroxy coumarin. After the copper(I)-catalyzed azide–alkyne cycloaddition reaction (‘click’ reaction) with alkyne-derivatized target molecules, the natively non-fluorescent construct has a strong enhancement in fluorescence. This fluorogenic dendrimer reporter can be used to efficiently monitor biological processes and the specificity afforded by the ‘click’ reaction greatly reduces background noise and enhances assay flexibility. We used this fluorogenic dendrimer reporter to monitor incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA, as a surrogate marker of cellular proliferation. We anticipate that this new class of fluorogenic reporter can be used to monitor a wide array of molecules and lends itself to high-throughput profiling of biological systems.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Screening, purification, and identification of a copper-dependent FITC-binding protein in human plasma: albumin

In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed "charcoal-based fluorescence assay" (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. This is the first to demonstrate that copper induces a covalent binding of FITC-labeled molecules with albumin. In addition, the developed CFA method facilitates the screening of small fluorescent dyes binding to macromolecules.     

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.     

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi- or trans-illuminators combined with photographic or charge-coupled device cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence

The fluorogenic properties of quercetin and similar flavonoids common in plants were exploited to analyse their interaction with target proteins. Quercetin produced a strong fluorescent signal upon binding to bovine serum albumin (BSA) and insulin. The fluorescent signal showed saturation kinetics with increasing flavonoid concentrations indicating the presence of defined peptide binding motifs. Other tested proteins showed no fluorescence with the flavonoids. In a comparative study including 22 flavonoids the compounds with fluorogenic properties were identified using our model proteins BSA and insulin and the structural requirements for the fluorogenic property were defined. Only flavones with a high degree of hydroxylation were able to elicit fluorescence. The emitted fluorescence was strongly enhanced at alkaline pH. Finally, an attempt was made to identify intracellular target molecules in live cells. Drosophila follicles showed a distinct staining pattern thus giving evidence that high concentrations of quercetin binding proteins are present in the nuclei and are associated with the ring canals. The presented biochemical and cytological data show that the interaction of the studied flavonoids with target proteins is specific and this finding opens up new experimental possibilities to systematically identify the cellular proteins with specific binding motifs for quercetin or other fluorogenic compounds of medical interest.     

Enzymic hydrolysis of intramolecular complexes for monitoring theophylline in homogeneous competitive protein-binding reactions

A novel approach in the design of fluorogenic substrate-analyte conjugates that can be used in a substrate-labeled fluorescent immunoassay (SLFIA) is described. The new SLFIA uses an enzyme substrate molecule that contains a fluorophore component and a quencher component, separated by a chain containing a bond which can be hydrolyzed by an enzyme. The feasibility of using this approach, in the construction of a fluorophore-quencher-analyte conjugate for monitoring analytes in homogeneous competitive protein binding reactions was demonstrated by using flavin-N⁶-(6-aminohexyl-theophylline) adenine dinucleotide (FAD-Theophylline) as the intramolecularly quenched fluorogenic substrate. Hydrolysis of the FAD-theophylline by nucleotide pyrophosphatase yielding FMN and AMP-theophylline restores the fluorescence to the expected level of FMN. Antibody to theophylline, however, inhibits the enzymic hydrolysis, and this inhibition is relieved in competitive binding when theophylline is added.     

Immobilized fluorogenic substrates for proteinases: a method for visualization and quantitation of elastase release from human monocytes

An elastase-specific fluorogenic substrate, 6-(N-carbo-benzoxy-L-alanyl-L-alanyl-L-alanylamido)-qu inoline, was synthesized and immobilized via the fluorophoric group to an alkylatable derivative of polyacrylamide microspheres. Upon hydrolysis by elastase, the proteolytic product of the reaction fluorescences with a characteristic greenish-yellow light corresponding to the presence of the 1-alkyl-6-aminoquinolinium ion. This method has been applied to detect the elastase activity released from monocytes grown on the microspheres. Because the fluorescent product is covalently attached to the microsphere and cannot diffuse away from the site of reaction, it is possible to identify individual cells releasing the proteinase molecules. These experiments demonstrate that covalently immobilized fluorogenic substrates can be used for direct visualization and quantitation of proteinase activity from individual cells in culture.     

A simple and sensitive enzyme-mediated assay of biotin.

Free biotin was quantitated by a competition by coating biotin-bovine serum albumin conjugate on a polystyrene microplate for binding to avidin-beta-galactosidase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by reaction with one of the following fluorogenic substrates, resorufin beta-D-galactoside and fluorescein di-beta-D-galactoside, in a fluorescence plate reader. Free biotin as little as 0.1 nmol can be routinely detected.     

Developing visible fluorogenic 'click-on' dyes for cellular imaging

New fluorogenic dyes were designed and synthesized based on Cu(I)-catalyzed 'click' reaction. Conjugating weakly fluorescent benzothiazole derivatives with an electron-deficient alkyne group at the 2-position with azide-containing molecules in aqueous solution form 'click-on' fluorescent adducts. Model reactions and cell culture experiment indicated that the developed 'click-on' dye could be applied to labeling various biomolecules, such as nucleic acids, proteins, and other molecules, in cells.     

用细菌/杆状病毒系统在昆虫细胞中表达GFP与HCV抗原融合蛋白

用细菌／杆状病毒（Ｂａｃ－ｔｏ－Ｂａｃ）系统在昆虫细胞中高效表达了绿色荧光蛋白（ＧＦＰ）与ＨＣＶ抗原的双功能融合蛋白,经ＥＬＩＳＡ测定和荧光显微镜观查证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＣＶ的抗原活性,实现了用绿色荧光蛋白等分子标记抗原,为免疫诊断新方法的建立打下了理论基础．     

Establishment of a suite of assays that support the discovery of proteasome stimulators

BackgroundThe proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing.MethodsThe cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates.ResultsTo demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold.ConclusionAssays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides.General significanceA suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.     

Fluorogenic substrates for lipases, esterases, and acylases using a TIM-mechanism for signal release

3-Acyloxyl-2-oxopropyl ethers of umbelliferone were investigated as new fluorogenic substrates for lipases and esterases. The aliphatic primary alcohol-leaving group released the fluorescent product umbelliferone by an enolization/beta-elimination reaction similar to the triose phosphate isomerase (TIM) reaction. A similarly designed phenylacetamide provided a fluorescent probe for penicillin G acylase, whereby the enolization/beta-elimination sequence from the intermediate aminoketone was very fast and spontaneous even under acidic conditions. The corresponding epoxyketone was not fluorogenic with epoxide hydrolases (EH). These substrates represent periodate-free Clips-otrade mark substrates.     

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA,protein and lipid

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein–ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2‐(2′‐hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein‐binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd.     

Controlling the diameter,monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry

Nanolipoprotein particles (NLPs) are nanometer‐scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein “scaffold,” which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell‐free protein synthesis followed by self‐assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo‐lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine “branches” are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell‐free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo‐EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6–30 nm) and polydispersity. The telodendrimer NLPs (TD‐NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.     

Let's think again about using mammalian temperature‐sensitive mutants to investigate functional molecules—The perspectives from the studies on three mutants showing chromosome instability

This review evaluates the use of temperature‐sensitive (ts) mutants to investigate functional molecules in mammalian cells. A series of studies were performed in which mammalian cells expressing functional molecules were isolated from ts mutants using complementation by the introduction and expression of the responsible protein tagged with the green fluorescent protein. The results showed that chromosome instability and cell‐cycle arrest were caused by ts defects in the following three molecules: the largest subunit of RNA polymerase II, a protein involved in splicing, and ubiquitin‐activating enzyme. The cells expressing functional protein were then isolated by introducing the responsible gene tagged with the green fluorescent protein to complement the ts phenotype. These cells proved to be useful in analyzing the dynamics of RNA polymerase II in living cells. Analyses of the functional interaction between proteins involved in splicing were also useful in the investigation of ts mutants and their derivatives. In addition, these cells demonstrated the functional localization of ubiquitin‐activating enzyme in the nucleus. Mammalian ts mutants continue to show great potential to aid in understanding the functions of the essential molecules in cells. Therefore, it is highly important that studies on the identification and characterization of the genes responsible for the phenotype of a mutant are carried out.     

A fluorescent probe for biothiols based on the conjugate addition of thiols to α,β‐unsaturated ester

A sensitive fluorogenic probe 1 for biothiols was developed based on the Michael addition reaction. The probe 1 was readily synthesized via the reaction of 2‐(4′‐hydroxyphenyl) benzimidazole (HPBI) with acryloyl chloride and shows weak fluorescence emission. Upon mixing with biothiols, the fluorescence of 1 is significantly enhanced due to the conjugate addition of thiols to the α,β‐unsaturated carbonyl moiety, thus eliminating the photoinduced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon–carbon double bond. Cysteine (Cys) was selected as the representative thiol in the spectral experiment. A good linear relationship was obtained from 1.0 to 30.0 µmol L^?1 for Cys and the detection limit was 0.17 µmol L^?1. Furthermore, probe 1 was highly selective for biothiols without the interference of some biologically relevant analytes and has been applied to detecting biothiols in human urine samples. Copyright © 2010 John Wiley & Sons, Ltd.