Time-resolved Fluorescence Anisotropy Research Articles

Nanoparticle Metrology of Silicates Using Time-Resolved Multiplexed Dye Fluorescence Anisotropy, Small Angle X-ray Scattering, and Molecular Dynamics Simulations.

We investigate the nanometrology of sub-nanometre particle sizes in industrially manufactured sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we investigate and quantify the advantages and limitations of multiplexing two fluorescent dye labels. Rotational times of the non-binding rhodamine B and adsorbing rhodamine 6G dyes are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to concentrations ranging between 0.2 M and 2 M of NaOH and on the stock solution at different temperatures. Additionally, it was shown that the particle size can also be measured using a single excitation wavelength when both dyes are present in the sample. The recovered average particle size has an upper limit of 7.0 ± 1.2 Å. The obtained results were further verified using small-angle X-ray scattering, with the recovered particle size equal to 6.50 ± 0.08 Å. To disclose the impact of the dye label on the measured complex size, we further investigated the adsorption state of rhodamine 6G on silica nanoparticles using molecular dynamics simulations, which showed that the size contribution is strongly impacted by the size of the nanoparticle of interest. In the case of the higher radius of curvature (less curved) of larger particles, the size contribution of the dye label is below 10%, while in the case of smaller and more curved particles, the contribution increases significantly, which also suggests that the particles of interest might not be perfectly spherical.

On-the-fly simulation of time-resolved fluorescence spectra and anisotropy.

We combine on-the-fly trajectory surface hopping simulations and the doorway-window representation of nonlinear optical response functions to create an efficient protocol for the evaluation of time- and frequency-resolved fluorescence (TFRF) spectra and anisotropies of the realistic polyatomic systems. This approach gives the effective description of the proper (e.g., experimental) pulse envelopes, laser field polarizations, and the proper orientational averaging of TFRF signals directly from the well-established on-the-fly nonadiabatic dynamic simulations without extra computational cost. To discuss the implementation details of the developed protocol, we chose cis-azobenzene as a prototype to simulate the time evolution of the TFRF spectra governed by its nonadiabatic dynamics. The results show that the TFRF is determined by the interplay of several key factors, i.e., decays of excited-state populations, evolution of the transition dipole moments along with the dynamic propagation, and scaling factor of the TFRF signals associated with the cube of emission frequency. This work not only provides an efficient and effective approach to simulate the TFRF and anisotropies of realistic polyatomic systems but also discusses the important relationship between the TFRF signals and the underlining nonadiabatic dynamics.

Insight into molecular interactions and energetics of surfactant self-assembly in hydrated glucose-urea deep eutectic solvent

Self-assembled structures in highly biocompatible Natural Deep Eutectic Solvent (NADES) are gaining popularity as new drug delivery systems due to their inherent advantages. Although glucose-urea (GU) based NADES satisfies the criterion of an efficient DES in terms of low toxicity, stability at desirable pH range, low viscosity and biocompatible polarity its potential as a medium for molecular self-assembly is yet unexplored. In this work we present first systematic study of self-assembly of pharmaceutically important excipients like sodium deoxycholate (NaDC), cetylpyridinium chloride (CPC) and Brij 35 (B35) in three hydrated GUNADES using isothermal titration calorimetry. The findings are correlated to the microstructure of these solvents as delineated from molecular dynamics simulations. Micro-environment of the micelles in a typical hydrated GU NADES has been studied using time resolved fluorescence anisotropy measurements. Cyto-toxicity of the proposed NADES has been assessed using MTT assay. CMC values of the surfactants increased with increase in glucose-urea concentration in the NADES indicating increased solvophilicity in GUNADES. Fluorescence studies indicate higher polydispersity of sodium deoxycholate in water than GUNADES. Cyto-toxicity study of GU80 shows good biocompatibility.

RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated.Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding.Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line.Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

Are "Carbon Dots" Always Carbon Dots? Evidence for their Supramolecular Nature from Structural and Dynamic Studies in Solution and in the Pure Solid.

A model for the morphology (size, shape, and crystallinity) of carbon dots (CDs) in the solid state consistent with the observed photoluminescence in solution is proposed herein. Overwhelming evidence has been collected that links the data coming from solid-state analysis (high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)) to that of solution (pulsed-field gradient (PFG)-NMR spectroscopy, time-resolved fluorescence anisotropy (TRFA), and steady-state/time-resolved fluorescence), allowing the establishment of an overall structural model for CDs. According to this model, the so-called carbon dots, observed under HRTEM imaging, are in fact supramolecular organized structures dynamically assembled from small to medium-sized molecular species when the solvent is removed to give the solid form. In this way, the imaged nanoparticles (TEM/AFM) are not covalently bound entities formed during the synthetic process, but instead supramolecular entities formed by noncovalent interactions. These particles, if at all present in solution, have the form of loose associations of relatively small molecules. This study was conducted on CDs obtained from the hydrothermal carbonization (HTC) of a biomass waste (olive wet pomace).

The Application of Fluorescence Anisotropy for Viscosity Measurements of Small Volume Biological Analytes.

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 μL and 100 μL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze-thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements' accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems.

Single-molecule FRET unmasks structural subpopulations and crucial molecular events during FUS low-complexity domain phase separation

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies permitting us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

Understanding the influence of ethylene glycol on the microscopic behavior of imidazolium-based monocationic and dicationic ionic liquid

In the present work, the influence of a molecular co-solvent, ethylene glycol (EG) on the microscopic behaviour of an imidazolium-based dicationic ionic liquid (DIL) is analysed by employing time-resolved fluorescence anisotropy (TRFA) and nuclear magnetic resonance (NMR) spectroscopic techniques. In order to have a clear understanding of the specific role of DIL, a mono-cationic ionic liquid (MIL) containing an alkyl side chain unit, same as the alkyl spacer chain length of DIL, has also been investigated. The rotational diffusion of two fluorescent probes 9-PA and R110 have been carried out in both neat MIL and DIL and their mixtures with various fractions of EG. The analysis of the results has demonstrated that, within the studied concentration range, the microstructure of MIL encompassing both polar and nonpolar domains gets disrupted as the mole fraction of EG increases. However, a similar investigation in DIL has revealed that the micro-structure of DIL gets affected slightly when similar proportions of EG are added to DIL. This behaviour of DIL is attributed to the enhanced rigidity of the DIL structure due to its folded structural arrangements. Furthermore, NMR studies have provided additional insights into the interactions between the components of ILs and the added solvent. It has been observed that EG can form strong hydrogen-bonding interactions with the imidazolium-ring and alkyl chain protons of MIL. However, such interaction has not been observed in DIL. Moreover, the translational diffusion coefficient (Dt) value for the cationic moiety of both MIL and DIL, measured through NMR, has depicted that in the presence of EG, the change in Dtvalue is much higher for MIL than that for DIL. Overall, the outcome of the combined fluorescence and NMR studies have pointed out that the behaviour of DIL and MIL in the presence of a co-solvent is very different from one another and thus the individual mixture (IL+solvent) can be used for various targeted industrial applications.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Does Viscosity Decoupling Guarantee Dynamic Heterogeneity? A Clue through an Excitation and Emission Wavelength-Dependent Time-Resolved Fluorescence Anisotropy Study.

Traditionally, deviation from Stokes-Einstein-Debye (SED) relation in terms of viscosity dependence of medium dynamics, i.e., with p ≠ 1, is taken as a signature of dynamic heterogeneity. However, it does not guarantee medium heterogeneity, as the decoupling may also originate from the deviation of the basic assumption of SED. Here, we developed a method to find a stronger relation between viscosity decoupling (p ≠ 1) and dynamic heterogeneity in terms of rotational motion. Our approach exploited the fact that in heterogeneous media, a solvatochromic probe will be solvated to a different extent at different microdomains (subpopulations), and photoselection of these subpopulations can be achieved by excitation or emission wavelength-dependent measurements. We hypothesized that the dynamics of a homogeneous system might show viscosity decoupling, but the extent of decoupling at different excitations (or at different emissions) should not be different. On the other hand, in a heterogeneous medium, this extent of viscosity decoupling (p-value) should be different at different excitations (or at different emissions). As proof of concept, we investigated three versatile solvent media: squalane (viscous molecular liquid), 1-ethyle-3-methylimidazolium ethyl sulfate ionic liquid (IL), and [0.78 acetamide + 0.22 LiNO3] deep eutectic solvent (DES). We found that squalane is homogeneous, although it shows fractional viscosity dependence (p ≠ 1). Interestingly, mild heterogeneity in IL and significant heterogeneity in the DES were observed. Overall, we conclude that the difference in the p-value as a function of excitation (or emission) wavelength-dependent might be a superior way for the detection of dynamic heterogeneity.

Probing Polarity and pH Sensitivity of Carbon Dots in Escherichia coli through Time-Resolved Fluorescence Analyses.

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2-8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM.

Role of Gemini Surfactants with Variable Spacers and SiO2 Nanoparticles in ct-DNA Compaction and Applications toward In Vitro/In Vivo Gene Delivery.

Compaction of calf thymus DNA (ct-DNA) by two cationic gemini surfactants, 12-4-12 and 12-8-12, in the absence and presence of negatively charged SiO2 nanoparticles (NPs) (∼100 nm) has been explored using various techniques. 12-8-12 having a longer hydrophobic spacer induces a greater extent of ct-DNA compaction than 12-4-12, which becomes more efficient with SiO2 NPs. While 50% ct-DNA compaction in the presence of SiO2 NPs occurs at ∼77 nM of 12-8-12 and ∼130 nM of 12-4-12, but a conventional counterpart surfactant, DTAB, does it at its concentration as high as ∼7 μM. Time-resolved fluorescence anisotropy measurements show changes in the rotational dynamics of a fluorescent probe, DAPI, and helix segments in the condensed DNA. Fluorescence lifetime data and ethidium bromide exclusion assays reveal the binding sites of surfactants to ct-DNA. 12-8-12 with SiO2 NPs has shown the highest cell viability (≥90%) and least cell death in the human embryonic kidney (HEK) 293 cell lines in contrast to the cell viability of ≤80% for DTAB. These results show that 12-8-12 with SiO2 NPs has the highest time and dose-dependent cytotoxicity compared to 12-8-12 and 12-4-12 in the murine breast cancer 4T1 cell line. Fluorescence microscopy and flow cytometry are performed for in vitro cellular uptake of YOYO-1-labeled ct-DNA with surfactants and SiO2 NPs using 4T1 cells after 3 and 6 h incubations. The in vivo tumor accumulation studies are carried out using a real-time in vivo imaging system after intravenous injection of the samples into 4T1 tumor-bearing mice. 12-8-12 with SiO2 has delivered the highest amount of ct-DNA in cells and tumors in a time-dependent manner. Thus, the application of a gemini surfactant with a hydrophobic spacer and SiO2 NPs in compacting and delivering ct-DNA to the tumor is proven, warranting its further exploration in nucleic acid therapy for cancer treatment.

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy.

Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, imaging is still rarely investigated in general and in carbon dots (CDs) in particular. To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and , together with the steady-state images of fluorescence intensity (FI) and FA (). The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. The FLT of fluorescein samples was found to decrease from to , whereas both and were significantly increased from to and to , respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of from to and from to for the first CDs and from to and to for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs. Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, , and ). Nevertheless, was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination

Time-resolved fluorescence anisotropy (FA) uses the fluorophore depolarization rate to report on rotational diffusion, conformation changes, and intermolecular interactions in solution. Although FA is a rapid, sensitive, and nondestructive tool for biomolecular interaction studies, the short (∼ns) fluorescence lifetime of typical dyes largely prevents the application of FA on larger macromolecular species and complexes. By using triplet shelving and recovery of optical excitation, we introduce optically activated delayed fluorescence anisotropy (OADFA) measurements using sequential two-photon excitation, effectively stretching fluorescence anisotropy measurement times from the nanosecond scale to hundreds of microseconds. We demonstrate this scheme for measuring slow depolarization processes of large macromolecular complexes, derive a quantitative rate model, and perform Monte Carlo simulations to describe the depolarization process of OADFA at the molecular level. This setup has great potential to enable future biomacromolecular and colloidal studies.

Cholesterol content regulates silica-induced lysosomal membrane permeability.

Inhalation of crystalline silica has been well documented to cause pulmonary inflammation and lung disease such as silicosis. Respirable silica particles deposit in the lungs and are phagocytosed by alveolar macrophages. Subsequently, phagocytosed silica remains undegraded within lysosomes causing lysosomal damage known as phagolysosomal membrane permeability (LMP). LMP can trigger the assembly of the NLRP3 inflammasome resulting in release of inflammatory cytokines that contribute to disease. In order to better understand the mechanisms of LMP this study used murine bone marrow derived macrophages (BMdM) as a cellular model to investigate the mechanism of silica-induced LMP. Reduction of lysosomal cholesterol in bone marrow derived macrophages with 18:1 phosphatidylglycerol (DOPG) liposome treatment increased silica-induced LMP and IL-1β release. Conversely, increasing lysosomal and cellular cholesterol with U18666A reduced IL-1β release. Co-treatment of bone marrow derived macrophages with 18:1 phosphatidylglycerol and U18666A resulted in a significant reduction of the effects of U18666A on lysosomal cholesterol. Phosphatidylcholine 100-nm liposome model systems were used to examine the effects of silica particles on lipid membrane order. Time-resolved fluorescence anisotropy of the membrane probe, Di-4-ANEPPDHQ, was used to determine changes to membrane order. Silica increased lipid order that was attenuated by inclusion of cholesterol in the phosphatidylcholine liposomes. These results demonstrate that increased cholesterol can attenuate silica-induced membrane changes in liposomes and cell models, while decreasing cholesterol exacerbates silica-induced membrane changes. Selective manipulation of lysosomal cholesterol may be a way of attenuating lysosomal disruption and preventing silica-induced chronic inflammatory disease progression.

Interfacial Structure and Electrostatics Related to Solute Activity in a Model Anionic-Surfactant/Polymer Self-Assembly.

Polymer/surfactant composites are used in industry as an excipient for water-insoluble solutes. Such enhanced dissolution ability of composite media is related to the spontaneous formation of pre-micellar polymer surfactant aggregates (PS) at a magnitude of order lower than the surfactant critical micelle concentration in water. Combining electrochemical and spectroscopic studies, we investigate the microscopic interfacial structure (i.e., interface electrostatics and surface polarity) of PS formed in composite media. We establish that in a composite system, a mere change in the polymer concentration at a fixed surfactant concentration makes possible to regulate the counter-ion binding ability, surface potential, surface charge density, packing and surface polarity of the PS interface. Our study shows that the higher dissolution of water-insoluble nonionic solutes in composite media is driven by the depressing of surface charge density and polarity of the PS interface. A similar modulation of the PS interface acts as a barrier for the passive relocation of water-soluble charged solutes into the PS pseudo-phase. The time-resolved fluorescence anisotropy study allows us to underline the effect of surface charge modulation on the dynamical aspects of solutes at the PS interface.

Using single-molecule fluorescence techniques to understand the enzymatic cycle of sex-biased RNA helicases DDX3X and DDX3Y.

Sex differences are pervasive in human health and disease with the most striking differences lying in the sex chromosomes, which encode a group of sex-specific protein homologs. DDX3X and DDX3Y are a pair of sexually dimorphic non-processive ATP-dependent RNA helicases. We previously showed that DDX3Y forms larger, less dynamic phase separated condensates compared to DDX3X, possibly due to the lower ATPase activity of DDX3Y and different dynamic interactions with RNA. We used single-molecule FRET of immobilized, fluorescently labeled RNA duplexes and reported that DDX3X and DDX3Y show different binding dynamics and partial unwinding activities (Shen and Yanas et al., Molecular Cell 2022). To further investigate the enzymatic activities of DDX3, we used multiparameter confocal spectroscopy to obtain unwinding, time-resolved anisotropy, diffusion coefficients, and structural changes by FRET of freely diffusing RNA duplexes upon addition of DDX3, with and without ATP. Duplex RNA with a 5’ overhang was labeled with a Cy3 and Alexa647 FRET pair at the chain termini. The proportion of duplex RNA was quantified and used as a readout for helicase activity, and DDX3X shows higher helicase activity than DDX3Y. With ns time-resolved fluorescence anisotropy, we found that the shorter, non-overhanging strand RNA is released with helicase activity, whereas protein remains bound to the longer RNA strand. Diffusion constants from fluorescence correlation spectroscopy revealed formation of enzymatically active protein clusters at low DDX concentrations. Removal of N- and C-terminal intrinsically disordered regions suppressed formation of clusters and reduced enzymatic activity. We hypothesize that these clusters act as “helicase hubs,” possibly tiny nuclei of liquid granules, which may be translational centers in the cell, leading to efficient translation initiation.

Ultrafast fluorescence depolarisation in green fluorescence protein tandem dimers as hydrophobic environment sensitive probes.

Advances in ultra-fast photonics have enabled monitoring of biochemical interactions on a sub nano-second time scale. In addition, picosecond dynamics of intermolecular energy transfer in fluorescent proteins has been observed. Here, we present the development of a genetically encoded fluorescent sensor that can detect changes in hydrophobicity by monitoring ultrafast fluorescence depolarisation. Our sensor is composed of a pair of dimeric enhanced green fluorescent proteins (dEGFPs) linked by a flexible amino-acid linker. We show dimerisation is perturbed by the addition of glycerol which interferes with the hydrophobic interaction of the two proteins. Time-resolved fluorescence anisotropy revealed a systematic attenuation of ultrafast fluorescence depolarisation when the sensor was exposed to increasing glycerol concentrations. This suggests that as hydrophobicity increases, dEGFP pairing decreases within a tandem dimer. Un-pairing of the protein fluorophores dramatically alters the rate of energy transfer between the proteins, resulting in an increase in the limiting anisotropy of the sensor.

Fluorescence of Bimolecular Guanine Quadruplexes: From Femtoseconds to Nanoseconds

The paper deals with the fluorescence of guanine quadruplexes (G4) formed by association of two DNA strands d(GGGGTTTTGGGG) in the presence of K+ cations, noted as OXY/K+ in reference to the protozoon Oxytricha nova, whose telomere contains TTTTGGGG repeats. They were studied by steady-state and time-resolved techniques, time-correlated single photon counting, and fluorescence upconversion. The maximum of the OXY/K+ fluorescence spectrum is located at 334 nm, and the quantum yield is 5.8 × 10-4. About 75% of the photons are emitted before 100 ps and stem from ππ* states, possibly with a small contribution of charge transfer. Time-resolved fluorescence anisotropy measurements indicate that ultrafast (<330 fs) excitation transfer, due to internal conversion among exciton states, is more efficient in OXY/K+ compared to previously studied G4 structures. This is attributed to the arrangement of the peripheral thymines in two diagonal loops with restricted mobility, facilitating the interaction among them and with guanines. Thymines should also be responsible for a weak intensity excimer/exciplex emission band, peaking at 445 nm. Finally, the longest living fluorescence component (∼2.1 ns) is observed at the blue side of the spectrum. So far, high-energy long-lived emitting states had been reported only for double-stranded structures but not for G4.

Probing Red Blood Cell Membrane Microviscosity Using Fluorescence Anisotropy Decay Curves of the Lipophilic Dye PKH26.

Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100-500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.