Very Bright Phycoerythrobilin Chromophore for Fluorescence Biolabeling

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells.     

A Tandem Green–Red Heterodimeric Fluorescent Protein with High FRET Efficiency

The tetrameric red fluorescent protein from Discosoma sp. coral (DsRed) has previously been engineered to produce dimeric and monomeric fluorescent variants with excitation and emission profiles that span the visible spectrum. The brightest of the effectively monomeric DsRed variants is tdTomato—a tandem fusion of a dimeric DsRed variant. Here we describe the engineering of brighter red (RRvT), green (GGvT), and green–red heterodimeric (GRvT) tdTomato variants. GRvT exhibited 99 % intramolecular FRET efficiency, resulting in long Stokes shift red fluorescence. These new variants could prove useful for multicolor live‐cell imaging applications.     

LSSmScarlet,dCyRFP2s,dCyOFP2s and CRISPRed2s,Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.     

One‐pot preparation of multicolor polymeric nanoparticles with high brightness by single wavelength excitation

Fluorescent nanoparticles with multiplex distinct emission signatures and high brightness by a single wavelength excitation are substantially needed in multiplex bioassays and imaging. In this study, we synthesized fluorescent polymeric nanoparticles incorporated with three polymerizable organic dyes via a one‐pot miniemulsion. By altering the doping ratio of three tandem dyes, the nanoparticles display abundant multiple fluorescence such as blue, cyan, green, orange, pink, red etc., together with distinguishable emission signatures under a single wavelength excitation, which were arising from the effective fluorescence resonance energy transfer (FRET) between the three energy‐matched dyes. Meanwhile, a large Stokes shift (up to 250 nm) can be generated by taking place multiple FRET cascade mechanism between donor and acceptor fluorophores in nanoparticles, which also suggests broad applications in biological labeling and imaging. Moreover, these nanoparticles are uniform in size, highly bright, excellently photostable, and shown prominent longterm stability. Overall, the novel multicolor fluorescent polymeric nanoparticles augur well for their potential applications in multiplexed bioanalysis and emitting displays. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41492.     

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.     

Functionalized Fluorescent Organic Nanoparticles Based AIE Enabling Effectively Targeting Cancer Cell Imaging

We report a fluorescent dye TM by incorporating the tetraphenylethylene (TPE) and cholesterol components into perylene bisimides (PBI) derivative. Fluorescence emission spectrum shows that the dye has stable red emission and aggregation-induced emission (AIE) characteristics. The incorporation of cholesterol components triggers TM to show induced chirality through supramolecular self-assembly. The cRGD-functionalized nanoparticles were prepared by encapsulating fluorescent dyes with amphiphilic polymer matrix. The functionalized fluorescent organic nanoparticles exhibit excellent biocompatibility, large Stokes’ shift and good photostability, which make them effective fluorescent probes for targeting cancer cells with high fluorescence contrast.     

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

In vivo expression of colored proteins without post‐translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell‐permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red‐shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2‐, 4‐, 5‐, 6‐, and 7‐azaindole) 4‐azaindole has exhibited the largest Stokes shift (～130 nm) in steady‐state fluorescence measurements. It is also highly biocompatible and as 4‐azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono‐ and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4‐azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole‐containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo‐ or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady‐state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.     

一种聚酰胺胺-苯甲醛树状大分子的合成及其荧光性能

首先以乙二胺为中心核,丙烯酸甲酯为支化单体合成了2.0代的聚酰胺胺树状大分子(PAMAM G 2.0),然后同苯甲醛在60℃水浴恒温反应48 h,得到了一种聚酰胺胺-苯甲醛树状大分子,用FTIR1、HNMR和13CNMR表征了合成产物的分子结构,结果和设计一致。该树状大分子能溶解于三氯甲烷,不溶于水、环己烷。对其荧光性能进行了研究,结果表明,溶液中的Fe3+对聚酰胺胺-苯甲醛树状大分子的荧光具有猝灭效应,并且荧光发射峰从436 nm红移到458 nm;溶液中的Zn2+对聚酰胺胺-苯甲醛树状大分子的荧光具有增强作用,同时荧光发射峰从436 nm蓝移到402 nm。     

Synthesis of Exosome-Based Fluorescent Gold Nanoclusters for Cellular Imaging Applications

In recent years, fluorescent metal nanoclusters have been used to develop bioimaging and sensing technology. Notably, protein-templated fluorescent gold nanoclusters (AuNCs) are attracting interest due to their excellent fluorescence properties and biocompatibility. Herein, we used an exosome template to synthesize AuNCs in an eco-friendly manner that required neither harsh conditions nor toxic chemicals. Specifically, we used a neutral (pH 7) and alkaline (pH 11.5) pH to synthesize two different exosome-based AuNCs (exo-AuNCs) with independent blue and red emission. Using field-emission scanning electron microscopy, energy dispersive X-ray microanalysis, nanoparticle tracking analysis, and X-ray photoelectron spectroscopy, we demonstrated that AuNCs were successfully formed in the exosomes. Red-emitting exo-AuNCs were found to have a larger Stokes shift and a stronger fluorescence intensity than the blue-emitting exo-AuNCs. Both exo-AuNCs were compatible with MCF-7 (human breast cancer), HeLa (human cervical cancer), and HT29 (human colon cancer) cells, although blue-emitting exo-AuNCs were cytotoxic at high concentrations (≥5 mg/mL). Red-emitting exo-AuNCs successfully stained the nucleus and were compatible with membrane-staining dyes. This is the first study to use exosomes to synthesize fluorescent nanomaterials for cellular imaging applications. As exosomes are naturally produced via secretion from almost all types of cell, the proposed method could serve as a strategy for low-cost production of versatile nanomaterials.     

Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.     

The mRubyFT Protein,Genetically Encoded Blue-to-Red Fluorescent Timer

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.     

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target—green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker—were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.     

New fluorescent monomers and polymers displaying an intramolecular proton‐transfer mechanism in the electronically excited state (ESIPT). IV. Synthesis of acryloylamide and diallylamino benzazole dyes and its copolymerization with MMA

Six new fluorescent monomers were synthesized by reaction of 2‐(5′‐amino‐2′‐hydroxyphenyl)benzazole derivatives with acryloyl chloride and allyl bromide. UV–vis and steady‐state fluorescence in solution were used to characterize its photophysical behavior. The monomers are fluorescent in the blue, green, yellow, and red region, with a large Stokes shift between 92 and 226 nm. A dual fluorescence ascribed to a conformational equilibrium in solution in the ground state dependent on the solvent polarity could be observed in the fluorescence emission spectra of the monomers. The radical polymerization of the monomers with methyl(methacrylate) allowed the production of fluorescent polymers in the blue–green region, with good optical and thermal properties. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci, 2006     

一种Hg~(2+)离子荧光探针的合成及荧光性质

以喹啉为原料,通过氧化、脱水、合环和还原作用合成了中间体5,10-二羟基苯并喹啉-6,9-二酮,后者再与氨基硫脲缩合合成终产物6-氧代-5,10-二羟基苯并喹啉-9-酮缩氨基硫脲。经1 H-NMR对产物进行结构表征,研究了终产物紫外吸收光谱、荧光光谱及其与各种离子配合后的光谱变化,结果表明其能与Hg2+选择性配位,确定其最大发射波长为534nm,Stokes位移为138nm。     

New fluorescent elastomeric materials based on synthetic and natural epoxidized rubbers

New fluorescent elastomeric materials were successfully prepared by reaction of an excited state intramolecular proton transfer-exhibiting silyl-functionalized benzothiazole dye with synthetic and natural epoxidized rubbers. The fluorescence emission and excitation spectra were obtained from the dye and the elastomeric materials to characterize its photophysical behavior. The benzothiazole derivative is fluorescent in the yellow region and presents a Stokes shift of 188 nm (solution) and 198 nm (solid state). After purification, the obtained materials from epoxidized rubbers presented excitation and emission maxima located at 358 nm and 550 nm, respectively, with a Stokes shift of 192 nm. The fluorescent dye could not be extracted from these films by solubilization–precipitation procedure, indicating the presence of covalent bonding between the dye and the matrix. On the other hand, the dye could be readily washed out of films that had been prepared using the corresponding nonepoxidized rubbers. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

氨基取代羟基苯基苯并咪唑衍生物的合成及荧光性质

合成了4种氨基取代的羟基苯基苯并咪唑类衍生物,并对它们的结构进行了表征,研究了取代基对于新化合物荧光性质的影响,实验证明:在羟基苯基苯并咪唑类化合物分子中引入氨基后,荧光发射波长发生了较大的红移(70~105nm),荧光斯托克斯位移显著增大(38~80nm),荧光量子收率有不同程度的减小。     

Identification of Potent Caspase-8 Inhibitors from a Library of Fluorescent Natural Products Screened by an AIEgen-Based Light-Up Probe

Fluorescent natural products are a rich source of drugs and chemical probes, but their innate fluorescence can interfere with fluorescence-based screening assays. Caspase-8 is a key player in apoptosis, its inhibition having been found to be beneficial for treatment of inflammatory and neurodegenerative diseases. Small-molecular inhibitors of caspase-8 remain sparsely reported, however. In this study, we firstly developed a light-up probe based on an AIEgen and capable of targeting caspase-8. This fluorescent dye has a Stokes shift of 200 nm, which could allow the innate fluorescence signals of natural products to be avoided. On screening a library of 86 fluorescent natural products, we found for the first time that gossypol showed potent inhibition of caspase-8 in vitro and in situ. This unique light-up probe, coupled with colored natural products, could represent an efficient approach to hit discovery for druggable targets.     

SNAP‐Tag‐Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides

Genetically encoded technologies provide methods for the specific labeling and imaging of proteins, which is essential to understand the subcellular localization of these proteins and their function. Herein, we employed naphthalimide, an efficient two‐photon fluorophore, to develop O⁶‐benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP‐tag. Three naphthalimide–BG derivatives, TNI‐BG, QNI‐BG, and ONI‐BG, were conveniently synthesized through modular “click chemistry” in high yields. All of them showed high labeling efficiency with SNAP‐tag in solution (≈1–2×10³ s^?1 m ^?1) and in bacteria. Among them, ONI‐BG showed high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP‐tag in mammalian cells. The protein‐labeled naphthalimides exhibited high two‐photon absorption cross‐sections, which indicated their potential application in protein‐specific two‐photon fluorescent imaging, such as two‐photon fluorescent lifetime imaging and two‐photon multicolor imaging. Therefore, ONI‐BG is a versatile tool that can be used to track subcellular proteins through multiple fluorescent techniques.     

Efficient phage display of intracellularly folded proteins mediated by the TAT pathway

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.     

Preparation and Characterization of Highly Fluorescent, Glutathione-coated Near Infrared Quantum Dots for in Vivo Fluorescence Imaging

Fluorescent probes that emit in the near-infrared (NIR, 700–1,300 nm) region are suitable as optical contrast agents for in vivo fluorescence imaging because of low scattering and absorption of the NIR light in tissues. Recently, NIR quantum dots (QDs) have become a new class of fluorescent materials that can be used for in vivo imaging. Compared with traditional organic fluorescent dyes, QDs have several unique advantages such as size- and composition-tunable emission, high brightness, narrow emission bands, large Stokes shifts, and high resistance to photobleaching. In this paper, we report a facile method for the preparation of highly fluorescent, water-soluble glutathione (GSH)-coated NIR QDs for in vivo imaging. GSH-coated NIR QDs (GSH-QDs) were prepared by surface modification of hydrophobic CdSeTe/CdS (core/shell) QDs. The hydrophobic surface of the CdSeTe/CdS QDs was exchanged with GSH in tetrahydrofuran-water. The resulting GSH-QDs were monodisperse particles and stable in PBS (phosphate buffered saline, pH = 7.4). The GSH-QDs (800 nm emission) were highly fluorescent in aqueous solutions (quantum yield = 22% in PBS buffer), and their hydrodynamic diameter was less than 10 nm, which is comparable to the size of proteins. The cellular uptake and viability for the GSH-QDs were examined using HeLa and HEK 293 cells. When the cells were incubated with aqueous solutions of the GSH-QDs (10 nM), the QDs were taken into the cells and distributed in the perinuclear region of both cells. After 12 hrs incubation of 4 nM of GSH-QDs, the viabilities of HeLa and HEK 293 cells were ca. 80 and 50%, respectively. As a biomedical utility of the GSH-QDs, in vivo NIR-fluorescence imaging of a lymph node in a mouse is presented.