Single-molecule Fluorescence Techniques Research Articles

A single CAA interrupt in a DNA three-way junction containing a CAG repeat hairpin results in parity-dependent trapping.

An increasing number of human disorders are attributed to genomic expansions of short tandem repeats (STRs). Secondary DNA structures formed by STRs are believed to play an important role in expansion, while the presence of nucleotide interruptions within the pure repeat sequence isknown to delay the onset and progression of disease. We have used two single-molecule fluorescence techniques to analyse the structure and dynamics of DNA three-way junctions (3WJs) containing CAG repeat hairpin slipouts, with and without a single CAA interrupt. For a 3WJ with a (CAG)10 slipout, the CAA interrupt is preferentially located in the hairpin loop, and the branch migration dynamics are 4-fold slower than for the 3WJ with a pure (CAG)10, and 3-fold slower than a 3WJ with a pure (CAG)40 repeat. The (CAG)11 3WJ with CAA interrupt adopts a conformation that places the interrupt in or near the hairpin loop, with similar dynamics to the pure (CAG)10 and (CAG)11 3WJs. We have shown that changing a single nucleotide (G to A)in a pure repeat can have a large impact on 3WJ structure and dynamics, which may be important for the protective role of interrupts in repeat expansion diseases.

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm2/s), slow (∼0.04 μm2/s), and fast (∼0.14 μm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

Revealing the heterogeneous catalytic kinetics of PtRu nanocatalysts at the single particle level.

Comparison of the structural features and catalytic performance of bimetallic nanocatalysts will help to develop a unified understanding of structure-reaction relationships. The single-molecule fluorescence technique was utilized to reveal the differences in catalytic kinetics among PtRu bimetallic nanocatalysts and Pt and Ru monometallic nanocatalysts at the single particle level. The results show that bimetallic nanocatalysts have higher apparent rate constants and desorption rate constants relative to monometallic nanocatalysts, which leads to their higher catalytic activity. At the single particle level, bimetallic nanocatalysts have a wider distribution of apparent rate constants, suggesting that bimetallic nanocatalysts have higher activity heterogeneity relative to monometallic nanocatalysts. By investigating the relationship between the reaction rate and the rate of dynamic activity fluctuations, it was found that spontaneous surface restructuring and reaction-induced surface restructuring of nanoparticles occurred. The surface of bimetallic nanoparticles restructured faster, which made the bimetallic nanocatalysts more active. These findings provide new insights into the design of highly active bimetallic nanocatalysts.

Single-Molecule Fluorescence Investigations of Solute Transport Dynamics in Nanostructured Membrane Separation Materials.

Many materials used for membrane separations are composed of nanoscale structures such as pores and domains. Such nanostructures often control the solute permeability and selectivity of the separation membranes. Thus, for future development of highly efficient separation membranes, it is important to understand the structural and chemical properties of these nanostructures and also their influences on solute transport dynamics. For the last two decades, single-molecule fluorescence techniques have been used to measure the detailed dynamics of solute molecules diffusing in various nanostructured materials, giving valuable insights into molecular transport mechanisms influenced by nanoscale material heterogeneity. This Perspective discusses recent single-molecule fluorescence studies on solute diffusion in materials relevant to membrane separations, including dense polymer films and nanoporous materials. These studies have revealed the formation and properties of nanostructures and unique transport dynamics of solute molecules manipulated by their confinement and partitioning to the nanostructures, which play key roles in membrane separations. This Perspective will also point out scientific challenges toward a thorough understanding of molecular-level mechanisms in membrane separations.

Strategies for Overcoming the Single-Molecule Concentration Barrier.

Fluorescence-based single-molecule approaches have helped revolutionize our understanding of chemical and biological mechanisms. Unfortunately, these methods are only suitable at low concentrations of fluorescent molecules so that single fluorescent species of interest can be successfully resolved beyond background signal. The application of these techniques has therefore been limited to high-affinity interactions despite most biological and chemical processes occurring at much higher reactant concentrations. Fortunately, recent methodological advances have demonstrated that this concentration barrier can indeed be broken, with techniques reaching concentrations as high as 1 mM. The goal of this Review is to discuss the challenges in performing single-molecule fluorescence techniques at high-concentration, offer applications in both biology and chemistry, and highlight the major milestones that shatter the concentration barrier. We also hope to inspire the widespread use of these techniques so we can begin exploring the new physical phenomena lying beyond this barrier.

New Confocal Microscope for Quantitative Fluorescence Lifetime Imaging with Improved Reproducibility

AbstractQuantitative single-molecule and time-resolved fluorescence techniques offer new insights into many samples in life and materials sciences. So far, their adoption has been slow because expert knowledge is required for correct data acquisition and analysis. Now, PicoQuant has developed a single-photon counting confocal microscope, called Luminosa. It combines state-of-the-art hardware with cutting-edge software to deliver high-quality data while simplifying daily operation. The system software includes context-based workflows for each technique, which improve accuracy, reproducibility, and quality of the data. This article showcases how this is achieved for fluorescence lifetime imaging (FLIM).

Dissecting diffusion and oligomerization dynamics of GPCRs in live cells using single-particle tracking.

Crucial insights about the signaling and trafficking mechanisms of G Protein Coupled Receptors (GPCRs) can be derived from their membrane diffusion properties. Previous studies in our lab (Shivnaraine et al., JACS 2016, Li at al., Biophys. J. 2018) have shown that the M2 muscarinic receptor (M2R) can be purified as oligomers, yet the oligomerization dynamics and the implications for signalling in live cells remain elusive. Here we used single-molecule fluorescence techniques, such as single-particle tracking (SPT) and single-molecule photobleaching (smPB), to characterize the dynamic distribution of M2R in HEK293 cells. To that end, receptors have been co-expressed at their N-terminus either with a green fluorescent protein (eGFP), or with a HaloTag for labelling with HaloTag ligand (HTL) dyes, such as JF549 HTL. For comparison, we applied the same techniques to μ-opioid receptors (MORs) in live U2OS cells. For both cases, measured diffusion maps are spatially and temporally heterogeneous, with receptors transitioning between normal and anomalous diffusion regimes. The analysis of experimental data was reinforced by simulations and by control measurements on monomeric (CD86) and dimeric (CD28) membrane proteins. Intensity traces of immobile, single receptor complexes and of monomeric/dimeric controls in the membrane of fixed cells were analyzed using an in-house smPB code based on a Bayesian algorithm (Garry et al., J. Chem. Phys. 2020). The results confirm the presence, correlated with expression levels, of higher order M2R oligomers in cells, and lay out the foundation for subsequent studies of their functional role.

Pairwise biosynthesis establishes ion channel balance and mitigates arrhythmia.

The coordinated expression of ion channels at the plasma membrane of cells underlies cardiac rhythms, neural signaling, cell cycle progression, and a host of other physiological processes involving every cell and tissue type in the body. Perturbation of this balance results in disease, yet the underlying mechanisms of physiological control are poorly understood. Here we examined ion channel expression from transcriptomics to translation. Using human myocardium and cardiomyocytes derived from induced pluripotent stem cells, we report that the expression of ion channel transcripts encoding components of the ventricular action potential is highly correlated. We showed that ion channels are synthesized as heterotypic pairs from discrete, interacting mRNA molecules through single-molecule fluorescence techniques and biochemical and electrophysiological approaches. Remarkably, targeted depletion of one species concomitantly reduced expression of the others and mitigated proarrhythmic behavior that would otherwise arise from the selective reduction of a single channel species. We propose that, on the background of coarse regulation of the transcriptome, pairwise mRNA assemblies, or microtranslatomes, confer stability through tandem biosynthesis and coregulation of electrically complementary ion channels mediating cardiac repolarization. Such assemblies may be a general mechanism determining the composition of macromolecular complexes where the quantitative balance of components is essential to function.

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.

Solvent-Induced Swelling Behaviors of Microphase-Separated Polystyrene-block-Poly(ethylene oxide) Thin Films Investigated Using In Situ Spectroscopic Ellipsometry and Single-Molecule Fluorescence Microscopy.

Block copolymers have attracted considerable interest in the fields of nanoscience and nanotechnology because these polymers afford well-defined nanostructures via self-assembly. An in-depth understanding of solvent effects on the physicochemical properties of these microdomains is crucial for their preparation and utilization. Herein, we employed in situ spectroscopic ellipsometry and single-molecule fluorescence techniques to gain detailed insights into microdomain properties in polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films exposed to ethanol- and water-saturated N2. We observed a quick increase and a subsequent gradual decrease in the ellipsometric thickness of PS-b-PEO films upon exposure to ethanol-saturated N2. This observation was unexpected because ethanol-saturated N2 induced negligible thickness change for PS and PEO homopolymer films. The similarity in maximum thickness gain observed under ethanol- and water-saturated N2 implied the swelling of PEO microdomains. Ethanol vapor permeation through the PEO microdomains was supported by the redshift of the ensemble and single-molecule fluorescence emission of Nile red in PS-b-PEO films. Single-molecule tracking data showed the initial enhancement and subsequent reduction of the diffusion of hydrophilic sulforhodamine B molecules in PS-b-PEO films upon exposure to ethanol-saturated N2, consistent with the spectroscopic ellipsometry results. The higher ethanol susceptibility of the PEO microdomains was attributable to their amorphous nature, as shown by FTIR data.

Ultraviolet optical horn antennas for label-free detection of single proteins

Single-molecule fluorescence techniques have revolutionized our ability to study proteins. However, the presence of a fluorescent label can alter the protein structure and/or modify its reaction with other species. To avoid the need for a fluorescent label, the intrinsic autofluorescence of proteins in the ultraviolet offers the benefits of fluorescence techniques without introducing the labelling drawbacks. Unfortunately, the low autofluorescence brightness of proteins has greatly challenged single molecule detection so far. Here we introduce optical horn antennas, a dedicated nanophotonic platform enabling the label-free detection of single proteins in the UV. This design combines fluorescence plasmonic enhancement, efficient collection up to 85° angle and background screening. We detect the UV autofluorescence from immobilized and diffusing single proteins, and monitor protein unfolding and dissociation upon denaturation. Optical horn antennas open up a unique and promising form of fluorescence spectroscopy to investigate single proteins in their native states in real time.

Bridging the gap between single molecule and bulk fluorescence techniques with PIE-FCCS

G protein-coupled receptors (GPCRs) are a diverse set of transmembrane proteins with large variations in sequence and domain topology. One important aspect of GPCR function is the role of spatial organization and dimerization/oligomerization, but there is still no systematic understanding of these effects. This is due, in part, to the techniques that have been used to analyze them. Bulk fluorescence techniques cannot fully quantify receptor concentration, diffusion and oligomerization state. Single molecule tracking techniques are excellent at determining membrane protein diffusion and protein-protein interactions, but the technology requires very low expression levels and so is challenging to perform at physiological expression levels.

Multiplexing single-molecule fluorescence measurements with spectroscopically diverse DNA-based FRET constructs

A fundamental challenge for single-molecule fluorescence techniques is simultaneous measurement of multiple analyte species with unique fluorescent labels. Although structurally different fluorophores could be used to create a set of labels with unique photophysical properties, they would also vary greatly in their chemical compatibility, and families of chemically similar fluorophores are limited in number. We have engineered a series of nanoscale DNA-FRET constructs that position the same donor-acceptor fluorophores (internal Cy3 / Cy5 integrated into the phosphate backbone) with different spacing and sequence contexts, which can be used as a large set of fluorescent labels to enable multiplexing of single-molecule fluorescence measurements.

Structural and compositional diversity in the kainate receptor family.

SUMMARYThe kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1–5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers.

Biology on track: single-molecule visualisation of protein dynamics on linear DNA substrates

Single-molecule fluorescence imaging techniques have become important tools in biological research to gain mechanistic insights into cellular processes. These tools provide unique access to the dynamic and stochastic behaviour of biomolecules. Single-molecule tools are ideally suited to study protein-DNA interactions in reactions reconstituted from purified proteins. The use of linear DNA substrates allows for the study of protein-DNA interactions with observation of the movement and behaviour of DNA-translocating proteins over long distances. Single-molecule studies using long linear DNA substrates have revealed unanticipated insights on the dynamics of multi-protein systems. In this review, we provide an overview of recent methodological advances, including the construction of linear DNA substrates. We highlight the versatility of these substrates by describing their application in different single-molecule fluorescence techniques, with a focus on in vitro reconstituted systems. We discuss insights from key experiments on DNA curtains, DNA-based molecular motor proteins, and multi-protein systems acting on DNA that relied on the use of long linear substrates and single-molecule visualisation. The quality and customisability of linear DNA substrates now allows the insertion of modifications, such as nucleosomes, to create conditions mimicking physiologically relevant crowding and complexity. Furthermore, the current technologies will allow future studies on the real-time visualisation of the interfaces between DNA maintenance processes such as replication and transcription.

Single Molecule Characterization of Amyloid Oligomers.

The misfolding and aggregation of polypeptide chains into β-sheet-rich amyloid fibrils is associated with a wide range of neurodegenerative diseases. Growing evidence indicates that the oligomeric intermediates populated in the early stages of amyloid formation rather than the mature fibrils are responsible for the cytotoxicity and pathology and are potentially therapeutic targets. However, due to the low-populated, transient, and heterogeneous nature of amyloid oligomers, they are hard to characterize by conventional bulk methods. The development of single molecule approaches provides a powerful toolkit for investigating these oligomeric intermediates as well as the complex process of amyloid aggregation at molecular resolution. In this review, we present an overview of recent progress in characterizing the oligomerization of amyloid proteins by single molecule fluorescence techniques, including single-molecule Förster resonance energy transfer (smFRET), fluorescence correlation spectroscopy (FCS), single-molecule photobleaching and super-resolution optical imaging. We discuss how these techniques have been applied to investigate the different aspects of amyloid oligomers and facilitate understanding of the mechanism of amyloid aggregation.

Single-Molecule Insights into Allosteric Regulation of the Nuclear Receptor Protein RXRα

Nuclear receptors are crucial transcription factors that respond to a broad range of external signaling ligands by binding cognate DNA response elements and switching on transcription. As such, these proteins form tightly regulated, multi-component allosteric systems. RXRα is one of the key players in this protein family for its role as a promiscuous heterodimerization partner. We employ single-molecule fluorescence techniques to investigate the conformational dynamics of RXRα's ligand binding domain (LBD)—the essential regulatory domain. It has been proposed that ligand binding changes the conformation of helix 12 of the LBD and positions it in a conformation favoring coactivator peptide binding. Our single-molecule FRET studies, however, demonstrate that helix 12 undergoes a substantial conformational change only after the coactivator peptide binds. This suggests an unexpected allosteric mechanism of nuclear receptor activation, which we investigate. In particular, we address the questions: on what time scales do the conformational changes of helix 12 take place? and how are its dynamics affected by modulations in other parts of RXRα and in its binding partners? Our studies will pave the way to better understanding of the complex regulatory machinery embodied in nuclear receptor function.

Single-Chain Dimensions of an Archetypal Phase-Separating Intrinsically Disordered Protein Region

Membrane-less organelles (MLOs) are non-stoichiometric assemblies of concentrated biomolecules lacking a surrounding lipid membrane and form via liquid-liquid phase separation (LLPS). They facilitate a variety of cellular processes, and the dysregulation of phase separation can lead to debilitating diseases. Intrinsically disordered regions (IDRs) in proteins have often been found to drive phase separation. Due to lack of structure, disordered proteins are challenging to study with traditional structural biology methods, and this combined with crowded dense phase environments requires careful experimental design in order to extract quantitative information. We are directing our studies at an archetypal IDR that undergoes homotypic phase separation, i.e. the prion-like domain (PLD) of hnRNPA1. Here we use single-molecule fluorescence techniques to explore the dimensions and dynamics of hnRNPA1 in light and dense phases. We characterize single-chain behavior in response to salt concentration, which is used to induce phase separation, and elucidate how this is modulated by sequence variation. Initial measurements show that the PLD is a compact disordered protein in the dilute regime and that modulation of the driving force for phase separation also modulates the single-chain dimensions. This work will provide insights into structural and dynamic features of phase-separating biomolecules and will enable the investigation of maturation effects responsible for disease states.

Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures.

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop structures during transcription. Using this technique, we found that R-loop formation precedes GQ formation and that there exists a positive feedback loop between G-quadruplex and R-loop formation.

Deciphering the photobehaviour of ensemble and single crystals of Zr-based ITQ MOF composites

Understanding the interactions that govern the photophysical behaviour of Metal-organic frameworks (MOFs) and their composites is paramount for their photochemical and photonic applications. In this work, we report on the photobehaviour of Nile Red (NR) adsorbed on the surface of 2D Zr-ITQ-HB (NR@Zr-ITQ-HB) MOF using ultrafast (fs-ns) and single-molecule fluorescence techniques. The results show the occurrence of energy transfer processes happening in 0.34 ns between neighbouring NR molecules, intramolecular charge transfer reaction (∼0.6 ps) and a vibrational cooling (10 ps) of the adsorbed dye. Time-resolved single-composite fluorescence microscopy experiments on different NR-loaded composites allowed to resolve the emission from adsorbed aggregates (0.9 ns) and from the charge separated state (2.5 ns) of excited NR. The different photobehaviour of NR adsorbed on ZrO2 nanoparticles indicates the relevance of the Zr-ITQ-HB MOFs surface to the observed photoinduced reactions in the composites. We discuss how the nature of both, the organic linkers and metal clusters in this kind of MOFs, affects the photodynamics of the composites. These results may help in designing MOFs "on demand" for their application in photons-based science and technology, like photocatalysis and lighting.