Changes In Fluorescence Anisotropy Research Articles

A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.

The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN)and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.

FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs.

With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time noninvasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here, we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and postimaging data analysis.

Effect of Ursolic and Oleanolic Acids on Lipid Membranes: Studies on MRSA and Models of Membranes.

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of life-threatening hospital- and community-acquired infections. A combination of antibiotics could be an opportunity to address the widespread emergence of antibiotic-resistant strains, including Methicillin-Resistant S. aureus (MRSA). We here investigated the potential synergy between ampicillin and plant-derived antibiotics (pentacyclic triterpenes, ursolic acid (UA) and oleanolic acid (OA)) towards MRSA (ATCC33591 and COL) and the mechanisms involved. We calculated the Fractional Inhibitory Concentration Index (FICI) and demonstrated synergy. We monitored fluorescence of Bodipy-TR-Cadaverin, propidium iodide and membrane potential-sensitive probe for determining the ability of UA and OA to bind to lipoteichoic acids (LTA), and to induce membrane permeabilization and depolarization, respectively. Both pentacyclic triterpenes were able to bind to LTA and to induce membrane permeabilization and depolarization in a dose-dependent fashion. These effects were not accompanied by significant changes in cellular concentration of pentacyclic triterpenes and/or ampicillin, suggesting an effect mediated through lipid membranes. We therefore focused on membranous effects induced by UA and OA, and we investigated on models of membranes, the role of specific lipids including phosphatidylglycerol and cardiolipin. The effect induced on membrane fluidity, permeability and ability to fuse were studied by determining changes in fluorescence anisotropy of DPH/generalized polarization of Laurdan, calcein release from liposomes, fluorescence dequenching of octadecyl-rhodamine B and liposome-size, respectively. Both UA and OA showed a dose-dependent effect with membrane rigidification, increase of membrane permeabilization and fusion. Except for the effect on membrane fluidity, the effect of UA was consistently higher compared with that obtained with OA, suggesting the role of methyl group position. All together the data demonstrated the potential role of compounds acting on lipid membranes for enhancing the activity of other antibiotics, like ampicillin and inducing synergy. Such combinations offer an opportunity to explore a larger antibiotic chemical space.

Simultaneous detection of reciprocal interactions between calmodulin, Ca2+ and molecular targets: a focus on the calmodulin-RyR2 complex.

In a recent issue of Biochemical Journal, Brohus et al. (Biochem. J.476, 193-209) investigated the interaction between the ubiquitous intracellular Ca2+-sensor calmodulin (CaM) and peptides that mimic different structural regions of the cardiac ryanodine receptor (RyR2) at different Ca2+ concentrations. For the purpose, a novel bidimensional titration assay based on changes in fluorescence anisotropy was designed. The study identified the CaM domains that selectively bind to a specific CaM-binding domain in RyR2 and demonstrated that the interaction occurs essentially under Ca2+-saturating conditions. This study provides an elegant and experimentally accessible framework for detailed molecular investigations of the emerging life-threatening arrhythmia diseases associated with mutations in the genes encoding CaM. Furthermore, by allowing the measurement of the equilibrium dissociation constant in a protein-protein complex as a function of [Ca2+], the methodology presented by Brohus et al. may have broad applicability to the study of Ca2+ signalling.

A Fluorescent Assay to Monitor Ligand-Dependent Closure of the Hexameric Rho Helicase Ring.

The bacterial Rho protein is an exemplar RecA-family hexameric helicase that assists with the termination of RNA polymerase activity on a variety of transcripts. During its catalytic cycle, Rho both loads onto and translocates along RNA through a series of tightly regulated, ligand-dependent conformational changes. Here we describe an assay to track Rho as it switches from an open-ring (RNA-loading) to a closed-ring (RNA-translocation) configuration by monitoring the association of a fluorescein-labeled RNA to Rho's central pore as a change in fluorescence anisotropy. The assay, which is in principle adaptable to the study of ligand-dependent isomerization events in other ring-shaped translocases, is readily amenable to 384-well format plates and small-molecule screening efforts.

A simple fluorescence anisotropy assay for detection of bisphenol A using fluorescently labeled aptamer

Bisphenol A (BPA) is one of the environmental endocrine disruptors (EDCs), and BPA contamination in environment can cause high risks to human health. Rapid determination of BPA on sites is in high demand in environmental analysis. Taking advantage of aptamers as affinity ligands and fluorescence anisotropy (FA) analysis, we developed a simple and rapid FA assay for BPA by employing a single tetramethylrhodamine (TMR) labeled short 35-mer DNA aptamer against BPA. The assay is based on the BPA-binding induced conformation change of TMR-labeled aptamer and alteration of interaction between TMR and guanine bases, resulting in change of FA signals. We screened the FA change of aptamer probes having TMR label on a specific site of the aptamer upon BPA addition. The aptamer with a TMR label on the 22nd T base showed large FA-decreasing response to BPA and maintained good binding affinity to BPA. By using this TMR-labeled aptamer, we achieved FA detection of BPA with a detection limit of 0.5 μmol/L under the optimized conditions. This assay was selective towards BPA and enabled the detection of BPA spiked in tap water sample, showing the potential applications on water samples.

Fluorescence Anisotropy-Based Signal-Off and Signal-On Aptamer Assays Using Lissamine Rhodamine B as a Label for Ochratoxin A.

Ochratoxin A (OTA), a common mycotoxin, has attracted great concern as many foodstuffs can suffer from OTA contamination; OTA causes harmful effects on human and animals. Rapid and sensitive detection of OTA is demanded in many fields for agricultural product quality, food safety, and health. Aptamer fluorescence polarization/anisotropy (FP/FA) assays integrate advantages of nucleic acid aptamers (e.g., easy preparation, high stability, and low cost) and FP/FA analysis (e.g., high sensitivity, rapidity, simplicity, and robustness). Here, we report the preparation of lissamine rhodamine B labeled OTA and developed competitive aptamer fluorescence anisotropy (FA) assays for OTA with signal-off or signal-on responses by using this fluorescently labeled probe. In the signal-off FA assay, the binding between the fluorescent probe and aptamer gave a large FA signal due to molecular volume increase, and the fluorescent probe was displaced from the aptamer in the presence of OTA target, causing FA to decrease. To further enhance the FA change in the signal-off assay, large-sized streptavidin was conjugated on the aptamer, and this assay allowed for a detection limit of 2.5 nM and a more remarkable FA decrease. Furthermore, we found that the fluorescent probe could interact with Tween 20, which caused the fluorescent probe to show a higher FA value than that of the aptamer-fluorescent probe complex. A signal-on FA assay was achieved in the binding buffer containing 0.1% Tween 20, with a detection limit of 10 nM. Signal-off and signal-on FA methods both were selective and enabled detection of OTA spiked in red wine samples, showing capability for target analysis in complex sample matrix.

Nucleotide-induced folding of cell division protein FtsZ from Staphylococcus aureus.

The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z-ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S.aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo-SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥1mm) dramatically shifts the equilibrium toward the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo-SaFtsZ also folds with 3.4m glycerol, enabling high-affinity GTP binding (KD 20nm determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N-oxide, and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ-GDP nucleotide-binding pocket that harbors different small compounds, which is available for extended nucleotide-replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichiacoli FtsZ, employing osmolyte-stabilized apo-FtsZs and the specific fluorescence anisotropy change in mant-GTP upon dissociation from the protein. This robust assay provides a basis to screening for high-affinity GTP-replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ-targeting antibacterial inhibitors.

Unexpected CK2β-antagonistic functionality of bisubstrate inhibitors targeting protein kinase CK2.

Protein kinase CK2, a heterotetrameric holoenzyme composed of two catalytic chains (CK2α) attached to a homodimer of regulatory subunits (CK2β), is a target for drug development for cancer therapy. Here, we describe the tetraiodobenzimidazole derivative ARC-3140, a bisubstrate inhibitor addressing the ATP site and the substrate-binding site of CK2 with extraordinary affinity (Ki=84pM). In a crystal structure of ARC-3140 in complex with CK2α, three copies of the inhibitor are visible, one of them at the CK2β interface of CK2α. Subsequent interaction studies based on microscale thermophoresis and fluorescence anisotropy changes revealed a significant impact of ARC-3140 and of its tetrabromo equivalent ARC-1502 on the CK2α/CK2β interaction. A structural inspection revealed that ARC-3140, unlike CK2β antagonists described so far, interferes with both sub-interfaces of the bipartite CK2α/CK2β interaction. Thus, ARC-3140 is a lead for the further development of highly effective compounds perturbating the quaternary structure of the CK2α2β2 holoenzyme.

Photophysics and photoreactivity of cross-conjugated enediynyl aggregates: Applications to multi-parametric sensing of microheterogeneity and reversible fluorescence switching

Fluorescent aggregates find wide-spread applications in modern optoelectronics. Here, we study the photophysics of organic fluorophores composed of benzene, pyrene, naphthalene, and anthracene moieties connected via a Y-shaped enediyne π-spacer in water-acetonitrile solvent mixtures and solid forms. The fluorophores show a predominant aggregate emission band, which is ca. 4000 cm−1 red-shifted compared to their locally excited (LE) emission energies in water-acetonitrile. The aggregate emission is however blue-shifted compared to an intramolecular charge transfer (ICT) emission band. The interplay of the LE, ICT, and aggregate emissive states depends on peripheral aromatic units, electronic substituents and microenvironments. The LE to aggregate emission spectral shift is exploited to sensitively probe micro-heterogeneity in bile salts, which are complemented by fluorescence intensity and fluorescence anisotropy changes. The solution phase aggregate emissions closely resemble the solid state aggregate emission bands, which are attributed to excimer states. The aggregate fluorescence states in the solids are altered by external stimuli and reversibly switched multiple of cycles. The aggregates undergo photoreactions in mixed-aqueous solvents, but they remain unreactive in solid powder forms.

Fluorescence Anisotropy Reduction of An Allosteric G-Rich Oligonucleotide for Specific Silver Ion and Cysteine Detection Based on the G-Ag+-G Base Pair.

Silver is a common heavy metal, and the detection of silver ion (Ag+) is of great importance because of its wide application and hazardous effect on the environment and human health. However, it is a great challenge to produce a large fluorescence anisotropy (FA) change for small molecules (e.g, Ag+). Herein, we describe a novel fluorescence anisotropy reduction approach for the sensitive and specific detection of Ag+. The feasibility of this method is demonstrated through screening a number of guanine-rich oligonucleotide probes. By selectively labeling the oligonucleotides with a single fluorophore tetramethylrhodamine (TMR), the reduction in FA response is associated with the conformation change from the unfolded to a hairpin-like folded structure by inducing formation of the intermolecular G-Ag+-G base pair, which diminishes the interaction between guanine and TMR by photoinduced electron transfer (PET). The change in FA allows the selective detection of Ag+ at a concentration as low as 0.5 nM with a dynamic range from 2.0 to 100 nM. The interference from the other 14 metal ions with a 100-fold even to a 1000-fold excess amount is negligible. This simple and cost-effective probe was further explored to determine cysteine (Cys) based on competing with a guanine-rich oligonucelotide for Ag+-binding.

Encapsulation and binding properties of curcumin in zein particles stabilized by Tween 20

This work studies the encapsulation and binding properties of curcumin in zein particles stabilized by Tween 20. The addition of Tween 20 is found to increase the encapsulation efficiency, stability, and radical scavenging ability of curcumin. As Tween 20 concentration (CTween 20) increases from 0 to 0.2 g/L, curcumin encapsulated in zein particles exhibits more pronounced increase in the absorption intensity than that at CTween 20 = 0.2–1.8 g/L, which is consistent with the changes of fluorescence intensity and anisotropy of curcumin. The encapsulation and spectroscopic properties of curcumin are explained by the bindings of curcumin in the hydrophobic cavities and the micellar like Tween 20 aggregates in zein particles, which are closely dependent on Tween 20 concentration. The binding constant results further suggest stronger binding affinity between curcumin and zein particles at CTween 20 above 0.2 g/L.

5-Carboxytetramethylrhodamine-Ampicillin Fluorescence Anisotropy-Based Assay of Escherichia coli Penicillin-Binding Protein 2 Transpeptidase Inhibition.

The high-molecular mass penicillin-binding proteins (PBPs) are the essential targets of the β-lactam classes of antibacterial drugs. In the Gram-negative pathogen Escherichia coli, these include PBP1a, PBP1b, PBP2, and PBP3. Techniques that enable facile measurement of the potency of inhibition of these targets are valuable for understanding structure-activity relationships in programs aimed at discovering new antibiotics to combat drug-resistant infections. Continuous fluorescence anisotropy-based assays for inhibition of soluble constructs of PBP1a, PBP2, and PBP3 from the serious Gram-negative bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii and PBP3 from E. coli using the fluorescent phenoxypenicillin analogue BOCILLIN FL have been described previously, but this technique was not useful for PBP2 from E. coli due to a lack of change in fluorescence anisotropy or intensity upon reaction. Here, we report that a fluorescent analogue of ampicillin, 5-carboxytetramethylrhodamine-ampicillin (5-TAMRA-ampicillin), was useful as the indicator in a continuous fluorescence anisotropy-based kinetic assay for inhibition of a soluble construct of PBP2 from E. coli. The assay enables measurement of the bimolecular rate constant for inhibition kinact /Ki. This measurement was made for representative drugs from four classes of β-lactams and for the diazabicyclooctenone ETX2514. 5-TAMRA-ampicillin was also useful in a fluorescence anisotropy-based assay for P. aeruginosa PBP2 and in fluorescence intensity-based assays with PBP1a and PBP3 from P. aeruginosa and A. baumannii and PBP3 from E. coli.

Constrained dynamics of the sole tryptophan in the third intracellular loop of the serotonin1A receptor

G protein-coupled receptors (GPCRs) are major signaling proteins in eukaryotic cells and are important drug targets. In spite of their role in GPCR function, the extramembranous regions of GPCRs are relatively less appreciated. The third intracellular loop (ICL3), which connects transmembrane helices V and VI, is important in this context since its crucial role in signaling has been documented for a number of GPCRs. Unfortunately, the structure of this loop is generally not visualized in x-ray crystallographic studies since this flexible loop is either stabilized using a monoclonal antibody or replaced with lysozyme. In this work, we expressed and purified the ICL3 region of the serotonin1A receptor and monitored its motional restriction and organization utilizing red edge excitation shift (REES) of its sole tryptophan and circular dichroism (CD) spectroscopy. Our results show that the tryptophan in ICL3 exhibits REES of 4 nm, implying that it is localized in a restricted microenvironment. These results are further supported by wavelength-selective changes in fluorescence anisotropy and lifetime. This constrained dynamics was relaxed upon denaturation of the peptide, thereby suggesting the involvement of the peptide secondary structure in the observed motional restriction, as evident from CD spectroscopy and apparent rotational correlation time. To the best of our knowledge, these results constitute one of the first measurements of motional constraint in the ICL3 region of GPCRs. Our results are relevant in the context of the reported intrinsically disordered nature of ICL3 and its role in providing functional diversity to GPCRs due to conformational plasticity.

A quantitative characterization of interaction between prion protein with nucleic acids

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.

Abstract 4029: Robust Ras inhibition assay utilizing fully synthetic Ras-binding domain

Abstract Intensifying efforts to develop clinically useful inhibitors of Ras proteins resulted in increasing need for quantitative, reliable and robust in vitro assays of Ras activity. The vast majority of detection methods used in the assays require fusion, chemical modification or immobilization of the protein. We have found that K-Ras modifications have significant impact on its activity and function because of the highly allosteric nature of the protein. We have attached three different fluorescent moieties to two sites distant from the active site of the protein in a site-selective manner. All six modifications resulted in significant drops in affinity towards Ras-binding domain of one of Ras's major effectors c-Raf as determined by Microscale Thermophoresis (MST). Fluorescent labeling of recombinant Raf1 RBD also resulted in inhibition of K-Ras binding. To produce a pair that interacted as unlabeled proteins, we have generated RBD with a single-site attached fluorescent moiety utilizing microwave-assisted high efficiency solid phase peptide synthesis. Optimization of coupling and deprotection protocols and the type of the solid support allowed conducting the synthesis of 76 residues long protein in fully-automated manner in one run. Refolding of the product yielded RBD that was indistinguishable from the recombinant one in CD spectra, melting temperature and K-Ras binding affinity as determined by isothermal calorimetry and microscale thermophoresis. Fluorescent RBD produced significant changes in both thermophoretic signal and fluorescence anisotropy upon binding to K-Ras. Both MST and fluorescence polarization assays have proven sensitive, reproducible and straightforward for determination of Ras inhibition constant for Ras inhibitors identified by virtual screens and rational design. They can be easily adapted to high throughput screening formats. Developed synthetic protocols allow for straightforward generation of proteins with various natural and non-natural modifications. Citation Format: Lyuba Khavrutskii, Sergey G. Tarasov, Colin Fields, Karen Stefanisko, Nadya Tarasova. Robust Ras inhibition assay utilizing fully synthetic Ras-binding domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4029. doi:10.1158/1538-7445.AM2017-4029

Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer

We developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5μM to 1mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer.

Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer

Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

Simultaneous imaging of multiple cellular events using high-accuracy fluorescence polarization microscopy.

Förster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules. To overcome this problem, we developed a 'homo-FRET'-based Ca2+ indicator, W-Cameleon, composed of two identical yellow fluorescent proteins. The binding of Ca2+ to the indicator induces a change in FRET efficiency, which in turn transforms into changes in fluorescence anisotropy. Given that the fluorescence polarization is depolarized by light passing through a high numerical aperture lens and reflecting on a dichroic mirror, we also developed a microscopy technique that reliably detects fluorescence anisotropy with high precision. Our design is aided by photonic-crystal technology, to compensate for the fluorescence depolarization. We thereby succeeded in the simultaneous visualization of three individual intracellular events by using three different fluorescent indicators. Our system may contribute to an expansion of the number of events that can be observed, which will enable a more quantitative understanding of biological phenomena.

Exonuclease III-assisted graphene oxide amplified fluorescence anisotropy strategy for ricin detection

Graphene oxide (GO) is an excellent fluorescence anisotropy (FA) amplifier. However, in the conventional GO amplified FA strategy, one target can only induce the FA change of one fluorophore on probe, which limits the detection sensitivity. Herein, we developed an exonuclease III (Exo III) aided GO amplified FA strategy by using aptamer as an recognition element and ricin B-chain as a proof-of-concept target. The aptamer was hybridized with a blocker sequence and linked onto the surface of magnetic beads (MBs). Upon the addition of ricin B-chain, blocker was released from the surface of MBs and hybridized with the dye-modified probe DNA on the surface of GO through the toehold-mediated strand exchange reaction. The formed blocker–probe DNA duplex triggered the Exo III-assisted cyclic signal amplification by repeating the hybridization and digestion of probe DNA, liberating the fluorophore with several nucleotides (low FA value). Thus, ricin B-chain could be sensitively detected by the significantly decreased FA. The linear range was from 1.0μg/mL to 13.3μg/mL and the limit of detection (LOD) was 400ng/mL. This method improved the sensitivity of FA assay and it could be generalized to any kind of target detection based on the use of an appropriate aptamer.