Fluorescent Förster Resonance Energy Transfer Research Articles

Tracking the Endosomal Escape of Nanoparticles in Live Cells Using a Triplex‐Forming Oligonucleotide

AbstractNanoparticle‐mediated intracellular delivery of oligonucleotides is a complex phenomenon that depends on the architecture and the intracellular trafficking of the engineered nanoparticles. Unravelling the molecular arrangements of oligonucleotides within the nanoparticles as well as their intracellular behavior are essential for designing effective nucleic acid delivery systems. Herein, a simple and general strategy for probing the endosomal escape of nanoparticles carrying oligonucleotides in live cells is reported. A triplex‐forming oligonucleotide probe is designed to target the transcription factor, kappa‐light‐chain‐enhancer of activated B cells (NF‐κB), in the cytosol of cells and to transduce the binding into a fluorescent Förster resonance energy transfer (FRET) signal. The combined use of the triplex‐forming oligonucleotide probe and super‐resolution microscopy enables the elucidation of the morphology, intracellular localization, and endosomal escape of the oligonucleotide‐loaded nanoparticles on a molecular level and with nanoscale resolution. The co‐delivery of the FRET probe and mRNA in cells via lipid‐ and polymer‐ based nanoparticles allow simultaneous correlation of the endosomal escape properties of nanoparticles and gene expression efficiency.

Small-Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications.

For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non-radiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore-quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

AbstractFor fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non‐radiatively, and are promising alternatives to fluorescent FRET acceptors. Small‐molecule (dark) quenchers have been widely used as acceptors in FRET‐based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small‐molecule quenchers that are used in FRET‐based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore–quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

Fluorescence-based techniques for the detection of the oligomeric status of proteins: implication in amyloidogenic diseases.

Intrinsically disordered proteins (IDPs) have captured attention in the last couple of decades due to their functional roles despite a lack of specific structure. Moreover, these proteins are found to be highly aggregation prone depending on the mutational and environmental changes to which they are subjected. The aggregation of such proteins either in the intracellular context or extracellular matrix is associated with several adverse pathophysiological conditions such as Alzheimer's, Parkinson's, and Huntington's diseases, Spinocerebellar ataxia, and Type-II diabetes. Interestingly, it has been noted that the smaller oligomers formed by IDPs are more toxic to cells than their larger aggregates. This necessitates the development of techniques that can detect the smaller oligomers formed by IDPs for diagnosis of such diseases during their early onset. Fluorescence-based spectroscopic and microscopic techniques are highly effective as compared to other techniques for the evaluation of protein oligomerization, organization, and dynamics. In this review, we discuss several fluorescence-based techniques including fluorescence/Förster resonance energy transfer (FRET), homo-FRET, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), fluorescence lifetime imaging (FLIM), and photobleaching image correlation spectroscopy (pbICS) that are routinely used to identify protein oligomers in extracellular and intracellular matrices.

Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells.

ABSTRACTCalpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.

FRET in a Polymeric Nanocarrier: IR-780 and IR-780-PDMS.

We introduce a method to monitor the integrity of micellar nanocarriers using a novel fluorescent dye, IR-780-PDMS and Förster resonance energy transfer (FRET) as a readout. In addition, these dye-loaded nanocarriers can be used as a phototoxic agent in vitro. Mainly, a nanocarrier was designed, based on a previously described amphiphilic ABA-copolymer (Pip-PMOXA-PDMS-PMOXA-Pip) scaffold that incorporates the fluorescent FRET dye partners IR-780-PDMS (donor) and IR-780 (acceptor). The confirmation of FRET (that only occurs when donor and acceptor are in the required close proximity of less than ∼10 nm) in the nanocarriers is used to prove that the acceptor dye (IR-780) is still contained in its hydrophobic core. We measured such FRET signals of the nanocarriers also upon cellular uptake into HeLa cells using fluorescence-lifetime imaging microscopy (FLIM). Confocal laser scanning microscopy after incubation with nanocarriers demonstrated the intracellular uptake of the particles and their localization in an intracellular granular pattern. To demonstrate the intactness of the nanocarriers by detection of FRET we measured the fluorescence lifetime (FLIM) of the donor dye. FLIM showed that both types of lifetimes, that of the quenched donor, and that of the unquenched donor were present, in a granular pattern and homogeneously in the cytosol, respectively, indicating the presence of intracellular intact and disintegrated micellar nanocarriers. These data show that the herewith-described FRET method allows monitoring the intactness of nanocarriers while en route to the target, and also that the cargo is delivered and released within a potential target cell. In addition, near-infrared (NIR) irradiation of IR-780-loaded micellar nanocarriers leads to photocytotoxicity, which we demonstrated in in vitro experiments. Our findings open potential avenues in photodynamic therapy (PDT) of cancer.

Graphene oxide based fluorescence super-quencher@QDs composite aptasensor for detection of Ricin B-chain

Graphene oxide (GO) has been widely used as a fluorescence quencher for constructing fluorescence sensing probes based on fluorescence Förster resonance energy transfer (FRET). To further improve its fluorescence quenching performance, we have introduced a water-soluble black hole quencher (BHQ-2) to be modified on the surface of carboxylated GO by chemical crosslinking method, and the fluorescence quenching properties of BHQ-2 modified GO nanocomposites (GO@BHQ-2) toward CdSe/ZnS QDs were analyzed in detail. Results show that the fluorescence quenching efficiency of GO@BHQ-2 as a dual energy acceptor, is significantly superior to that of unmodified GO. Therefore, GO@BHQ-2 can be used as a fluorescence super-quencher for designing highly sensitive biological fluorescent probes. Moreover, the GO@BHQ-2 nanocomposites were further applied to the construction of QD-based aptasensor for the quantitative detection of ricin B-chain (RTB). The established probe can detect RTB in the range of 0.05–3.0 μg mL−1 (R2 = 0.991), with a detection limit of 0.037 μg mL−1. The proposed probe can also be used to detect RTB in real samples, and thus it has potential applications in biological analysis.

Intrabody-based FRET probe to visualize endogenous histone acetylation

Post-translational histone modifications are major regulators of gene expression. However, conventional immunoassays do not provide sufficient information regarding their spatial and temporal dynamic changes. Fluorescence/Förster resonance energy transfer (FRET)-based probes are capable of monitoring the dynamic changes associated with histone modifications in real-time by measuring the balance between histone-modifying enzyme activities. Recently, a genetically encoded histone-modification fluorescent probe using a single-chain variable region (scFv) fragment of a specific antibody was developed. The probe, modification-specific intracellular antibody, is capable of monitoring histone-acetylation levels in both cultured cells and living organisms based on the ratio of fluorescence intensities between the cell nucleus and cytoplasm. In this study, we constructed a FRET probe composed of yellow fluorescent protein attached at the N-terminus of an acetyl H3K9-specific scFv, tethered to a cyan fluorescent protein. When the FRET probe was expressed in human cells, both FRET efficiency and fluorescence intensity in the nucleus increased following histone-deacetylase inhibitor treatment. Using these two parameters, endogenous histone-acetylation levels were quantified over a high dynamic range. This probe provides a simple approach to quantify spatial and temporal dynamic changes in histone acetylation.

Familial Alzheimer’s disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer’s disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear.To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway.Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD.

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

Abstract The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.

Ion-Selective Optical Nanosensors Based on Solvatochromic Dyes of Different Lipophilicity: From Bulk Partitioning to Interfacial Accumulation.

The sensing mechanism of fluorescent ion-selective nanosensors incorporating solvatochromic dyes (SDs), with K+ as model ion, is shown to change as a function of dye lipophilicity. Water-soluble SDs obey bulk partitioning principles where the sensor response directly depends on the lipophilicity of the SD and exhibits an influence on the phase volume ratio of nanosensors to aqueous solution (dilution effect). A lipophilization of the SDs is shown to overcome these limitations. An interfacial accumulation mechanism is proposed and confirmed with Förster resonance energy transfer (FRET) with a ratiometric near-infrared fluorescence FRET pair. This work lays the foundation for operationally more robust ion-selective nanosensors incorporating SDs.

Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.

A novel role of IκBα and WWOX in promoting lymphoid Molt-4 and Jurkat T cell differentiation (36.42)

Abstract Human common fragile site gene WWOX, encoding WW domain-containing oxidoreductase (designated WWOX, FOR or WOX1), plays critical roles in the pathogenesis of cancer and maintenance of normal physiology. To explore the role of WOX1 in lymphoid cancer, here we determined that transient expression of WOX1 induced the expression of a differentiation marker CD3 in an acute lymphoblastic leukemia cell line Molt-4, but the induction was less effective in better-differentiated Jurkat T cells. Upregulation of RORγ, one of the Th17 cell markers, was shown in Molt-4, but not Jurkat T cells, overexpressing WOX1. Mapping analysis revealed that the N-terminal WW domain of WOX1 induced T lymphoid cell differentiation, in which a dominant negative and phosphorylation mutants (i.e. Y33R and Y61R) of WOX1 blocked the effect. We determined that the N-terminal WW domain of WOX1 physically interacts with IκBα (an inhibitor of NF-κB), as determined by co-immunoprecipitaiton and Fluorescence/Förster resonance energy transfer (FRET). Ectopic IκBα, via its N-terminal ankyrin-repeat region, induced Molt-4 differentiation. Both WOX1 and IκBα did not enhance T cell differentiation synergistically, suggesting that they may nullify each other’s function. In positive controls, phorbol and ionophore A23187 induced cell differentiation by upregulating IκBα, CD3 and WOX1 in Molt-4 T-cells. Together, our finding demonstrates a novel WOX1/IκBα regulatory mechanism for T-cell differentiation.

Towards a FRET-based immunosensor for continuous carbohydrate monitoring

In this report we have evaluated the potential of using fluorescence/Förster resonance energy transfer (FRET) in a competitive immunosensor for continuous monitoring of the carbohydrate hapten maltose. The cyanine dyes Cy5 and Cy5.5 were used as a donor–acceptor pair by conjugation to maltose-labeled bovine serum albumin (BSA) and the monoclonal antibody IgG 39.5, giving Cy5–BSA–maltotriitol (3.1/1/18) and Cy5.5–mAb39.5 (2.2/1), respectively. This antibody with weak affinity towards maltose showed full reversibility to both the free maltose and the maltose-labeled conjugate. It allowed us to measure continuously the maltose content by monitoring the FRET signal change over time due to displacement of Cy5–BSA–maltotriitol from Cy5.5–mAb39.5 inside a semipermeable capsule. A near 22% total increase was seen in the fluorescence intensity ratio I 670/ I 700 in the presence of maltose, with a calculated EC 50 = 1.87 ± 0.13 mM ( R 2 = 0.9984) from the sigmoidal dose–response curve at 25 °C. Specificity of the immunosensor was shown with the structural analog to maltose, cellobiose, and it generated no detectable response. A minor drift in the sensor baseline was seen with 0.4% per 24 h, which was in the same magnitude as the signal-to-noise ratio, during the 4 weeks of measurements. The immunosensor was applied to crude samples of oat drinks for direct quantification of the maltose content. Overall, this work demonstrates the potential to use an immunosensor based on weakly binding antibodies and FRET technology for remote and non-invasive carbohydrate monitoring.

Picosecond-resolution fluorescence lifetime imaging microscopy: a useful tool for sensing molecular interactions in vivo via FRET

Fluorescence lifetime imaging microscopy (FLIM) provides a promising, robust method of detecting molecular interaction not not nots in vivo via fluorescence/Förster resonance energy transfer (FRET), by monitoring the variation of donor fluorescence lifetime, which is insensitive to many artifacts influencing convential intensity-based measurements, e.g. fluorophore concentration, photobleaching, and spectral bleed-through. As proof of principle, we demonstrate the capability of a novel picosecond-resolution FLIM system to detect molecular interactions in a well-established FRET assay. We then apply the FLIM system to detect the molecular interaction of a transforming oncogene RhoC with a binding partner RhoGDIgamma in vivo, which is critical to understand and interfere with Rho signaling for cancer therapeutics.