Single Binding Event Research Articles

Measuring Stepwise Binding of Thermally Fluctuating Particles to Cell Membranes without Fluorescence

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here, we show that it is possible to detect single binding events of immunoglobulin-G-coated polystyrene beads, which are held in an optical trap near the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically, and no fluorophore labeling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential stepwise increases in the force constant of the bond between a bead and a cell of typically 20 pN/μm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes, including phagocytosis, drug delivery, and the effects of small microplastics and particulates on cells.

Molecular Mechanisms for the Stochastic Condensation of LAT Assemblies in T Cells

T cells discriminate between self and agonist by requiring long dwelling binding events between peptide-MHC (pMHC) and the T Cell Receptor (TCR). A consequence of successful signal transduction is the formation of a multi-molecular assembly of downstream molecules that is crosslinked by the phosphorylated scaffold protein Linker for Activation of T cells (LAT). While it is known that after ligand binding Zap70 kinase recruits to the TCR and begins phosphorylating LAT, the early molecular events precipitating LAT condensation in relation to a single ligated TCR is unknown. Using supported lipid bilayer technology and TIRF microscopy, we directly measure the intracellular responses to single pMHC-TCR binding events in living primary T cells with high spatiotemporal, multi-color resolution. This provides the ability to accurately determine the order of events in TCR signal transduction in relation to a single TCR, as opposed to bulk stimulation methods. We observe that after a pMHC binds to a TCR that LAT condensation occurs after an extended delay. While this timing is likely set, in part, by a competition of kinase/phosphatase pressure and LAT diffusion, the phosphorylation of LAT has a similarly extended delay. This suggests that diffusive phospho-LAT requires an additional event to trigger aggregation. These data demonstrate novel roles for key signaling proteins and, in contrast to ensemble methods, are the first to visualize the decisive steps leading to LAT condensation and completion of kinetic proofreading.

The Formation of Lat Protein Condensates in Response to Single pMHC-TCR Binding Events

The Formation of Lat Protein Condensates in Response to Single pMHC-TCR Binding Events

Amphiphilic ligand modified gold nanocarriers to amplify lanthanide loading for ultrasensitive DELFIA detection of Cronobacter.

Conventional dissociation-enhanced lanthanide (Ln3+) fluoroimmunoassays (DELFIAs) using Ln3+ chelate-labeled antibodies as molecular probes exhibit limited sensitivity because of their relatively low Ln3+ labeling ratio per biomolecule. Herein, we applied gold nanoflowers (AuNFs) as amplified nanocarriers to increase the Ln3+ labeling ratio in a single molecular binding event for improving the sensitivity of traditional DELFIA. Two thiolated amphiphilic ligands (thiolated ethylenediaminetetraacetic acid (EDTA) and thiolated acylhydrazine-terminated ligands), consisting of a hydrophobic alkane chain, oligo(ethylene glycol) unit, and functional terminal of the EDTA or acylhydrazine group, were designed for the surface modification of AuNFs. The resultant ligand-coated AuNFs exhibited dual functions of Ln3+ chelation via the EDTA group and oriented attachment of antibodies via the acylhydrazine group. By utilizing 80 nm AuNFs as amplified carriers, we demonstrated that the maximum Eu3+ loading amount reached 1.07 × 104 Eu3+ ions per AuNF, which is approximately two to three orders of magnitude higher than that of traditional molecular probes, thereby amplifying the luminescence signal and enhancing the sensitivity of DELFIA. By combining a magnetic-mediated sandwich-type DELFIA method, the designed amplified AuNF nanoprobes achieved an ultrasensitive luminescence detection of Cronobacter muytjensii with a limit of detection (LOD) of 1.2 × 102 cfu mL-1 in a powdered infant formula. This LOD value was ca. 230-fold lower than that of the traditional colorimetric immunoassay. The designed signal amplification strategy using bifunctional ligand-modified AuNFs as enhanced Ln3+ nanocarriers provided a huge potential for building various ultrasensitive luminescence immunoassays for in vitro biodetection.

Discrimination between target and non-target interactions on the viral surface by merging fluorescence emission into Rayleigh scattering.

Direct and quantitative determination of antibodies or cellular receptors dynamically binding to the surface of viral particles is the key issue for predicting the efficacy of therapeutic materials or host susceptibility to a new emerging pathogen. However, targeted visualization of infectious viruses is still highly challenging owing to their nanoscopic sizes and uncontrollable nonspecific interactions with loading molecules responsible for false signals. Here we present a multimodal single-molecule and single-particle (SMSP) visualization capable of simultaneously yet independently tracking Rayleigh scattering and fluorescence that, respectively, are generated from viruses (approximately 100 nm) and labeled interacting molecules. By analyzing real-time trajectories of fluorescent antibodies against a virus surface protein with reference to single virus-derived Rayleigh scattering, we determined heterogeneous binding stoichiometry of virus-antibody couplings irrespective of the nonspecific binder population. Therefore, our multimodal (or multi-level) SMSP assay visually identifies and selectively quantifies specific interactions between them with single binding event accuracy. As a 'specific-binding quantifier' to assess variable host susceptibility to a virus, it was further applied for distinguishing ratiometric bindings and spontaneous dissociation kinetics of synthesized isomeric receptors to influenza virus. The present framework could offer a solid analytical foundation for the development of a direct-acting antiviral agent inhibiting an integral viral enveloped protein and for nanobiological investigation for dissecting spatiotemporal nanoparticle-molecule interactions, which have been scarcely explored compared to those among plasmonic nanoparticles or among molecules only.

Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å.

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5′-GCG↓C-3′ in duplex DNA and cleaves (‘↓’) to produce fragments with 2-base, 3′-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the ‘PD-D/EXK’ superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5′-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.

Giant single molecule chemistry events observed from a tetrachloroaurate(III) embedded Mycobacterium smegmatis porin A nanopore

Biological nanopores are capable of resolving small analytes down to a monoatomic ion. In this research, tetrachloroaurate(III), a polyatomic ion, is discovered to bind to the methionine residue (M113) of a wild-type α-hemolysin by reversible Au(III)-thioether coordination. However, the cylindrical pore geometry of α-hemolysin generates shallow ionic binding events (~5–6 pA) and may have introduced other undesired interactions. Inspired by nanopore sequencing, a Mycobacterium smegmatis porin A (MspA) nanopore, which possesses a conical pore geometry, is mutated to bind tetrachloroaurate(III). Subsequently, further amplified blockage events (up to ~55 pA) are observed, which report the largest single ion binding event from a nanopore measurement. By taking the embedded Au(III) as an atomic bridge, the MspA nanopore is enabled to discriminate between different biothiols from single molecule readouts. These phenomena suggest that MspA is advantageous for single molecule chemistry investigations and has applications as a hybrid biological nanopore with atomic adaptors.

Detecting and Directing Single Molecule Binding Events on H-Si(100) with Application to Ultradense Data Storage.

Many diverse material systems are being explored to enable smaller, more capable and energy efficient devices. These bottom up approaches for atomic and molecular electronics, quantum computation, and data storage all rely on a well-developed understanding of materials at the atomic scale. Here, we report a versatile scanning tunneling microscope (STM) charge characterization technique, which reduces the influence of the typically perturbative STM tip field, to develop this understanding even further. Using this technique, we can now observe single molecule binding events to atomically defined reactive sites (fabricated on a hydrogen-terminated silicon surface) through electronic detection. We then developed a simplified error correction tool for automated hydrogen lithography, quickly directing molecular hydrogen binding events using these sites to precisely repassivate surface dangling bonds (without the use of a scanned probe). We additionally incorporated this molecular repassivation technique as the primary rewriting mechanism in ultradense atomic data storage designs (0.88 petabits per in2).

Probing Single Molecule Binding and Free Energy Profile with Plasmonic Imaging of Nanoparticles.

Measuring binding between molecules is critical for understanding basic biochemical processes, developing molecular diagnosis, and screening drugs. Here we study molecular binding at the single molecule level by attaching nanoparticles to the molecular binding pairs. We track the thermal fluctuations of the individual nanoparticles with sub-nanometer precision using a plasmonic scattering imaging technique and show that the fluctuations are controlled by the molecular binding pairs rather than by the nanoparticles. Analysis of the thermal fluctuations provides unique information on molecular binding, including binding energy profile, effective spring constant, and switching between single and multiple molecular binding events. The method provides new insights into molecular binding and also allows one to differentiate nonspecific binding from specific binding, which has been a difficult task in biosensors.

Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.

Beyond the TCR, Antigen Discrimination in T Cells Continues in the LAT:GRB2:SOS Protein Condensate

T cells activate in response to a stochastic series of single molecule binding events between T cell receptors (TCRs) and peptide ligands presented on major histocompatibility complexes (pMHCs) accumulated at a cell-cell interface. Discrimination between ligands with subtly different affinities ensures selective and robust immunological responses upon TCR triggering. Formation of protein condensates based on the scaffolding protein Linker for Activation of T cells (LAT) is a key intermediate step in TCR signaling. LAT is multi-phosphorylated on tyrosine residues by the kinase, ZAP70, which is activated by triggered TCRs. The phosphotyrosines recruit Grb2, which, via interactions with the multivalent GEF, SOS, forms an extended LAT:Grb2:SOS assembly that resembles a phase transition. We mapped single pMHC:TCR binding events in T cells expressing fluorescent protein-tagged LAT and show that LAT assemblies are discrete and have characteristic features that are independent of the originating binding event. Furthermore, LAT assemblies can become spatially uncoupled from the pMHC:TCR complex. These individual assemblies are capable of deforming the plasma membrane and creating local domains that may participate in protein segregation or control reaction kinetics. The probability of LAT assembly is a function of the pMHC affinity, suggesting a second layer of kinetic proofreading that exists beyond the triggered TCR. We recently introduced a molecular impulse-response assay which maps pMHC:TCR binding events to early T cell activation. Results here argue that LAT assemblies may confer a molecular memory that connects a series of spatiotemporally correlated binding events to cellular decision-making. By extending the impulse-response assay, we show that activating cells experience a characteristic, fast rate of LAT assembly. Taken together, we present new molecular dimensions to T cell activation that could provide actionable therapeutic insights.

Flow-Induced Dispersion Analysis (FIDA) for Protein Quantification and Characterization.

Flow-Induced Dispersion Analysis (FIDA) enables characterization and quantification of proteins under native conditions. FIDA is based on measuring the change in size of a ligand as it selectively interacts with the target protein. The unbound ligand has a relatively small apparent hydrodynamic radius (size), which increase in the presence of the analyte due to binding to the analyte. The Kd of the interaction may be obtained in a titration experiment and the measurement of the apparent ligand size in an unknown sample forms the basis for determining the analyte concentration. The apparent molecular size is measured by Taylor dispersion analysis (TDA) in fused silica capillary capillaries. FIDA is a "ligand-binding" assay and has therefore certain features in common with Enzyme-Linked Immunosorbent Assay (ELISA), Surface Plasmon Resonance (SPR), and Biolayer Interferometry (BLI) based techniques. However, FIDA probes a single in-solution binding event and thus makes assay development straightforward, and the absolute size measurement enables built-in assay quality control. Further, as FIDA does not involve surface chemistries, complications related to nonspecific adsorption of analyte and assay components are minimized enabling direct measurement in, e.g., plasma and serum.

Ultrasensitive Detection of DNA Based on Exonuclease III-Assisted Recycling Amplification and DNAzyme Motor.

Herein, we have developed a dual amplification strategy for ultrasensitive detection of DNA combining exonuclease III (Exo III)-assisted reaction and DNAzyme motor. DNA probes are carefully designed; thus, target recognition and the first amplification cycle are accomplished simultaneously, which makes the operation very convenient. Moreover, the self-powered DNAzyme motor may translate a single binding event into cleavage of multiple fluorescence probes, which significantly heightens the signal intensity. As a result, the limit of detection as low as 21 fM is achieved. The fluorescence intensity is found to have a linear relationship with respect to the logarithm of DNA concentration in a wide range from 100 fM to 10 nM. This proposed method shows great potential for the applications of biological studies and clinical diagnosis.

(Invited) Near Infrared Chemical Imaging of Cellular Communication Using Carbon Nanotubes

Intercellular chemical communication proceeds with both spatial and temporal components. A very prominent example is neurotransmitter release from neurons. But so far there are no analytical tools available to detect release and propagation of signaling molecules on small length scales and fast time scales. We have developed fluorescent nanosensors to monitor interactions of cells with their environment e.g. for chemical imaging of small molecules released by cells. For that purpose we used near infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) and chemically rendered them selective for small signaling molecules such as the neurotransmitter dopamine. When dopamine binds the quantum yield of the sensors increased by up to 400 % and we were able to report single dopamine binding events. Parallel imaging of many of those sensors provides a nIR image (900 nm -1300 nm) of the dopamine concentration around cells. Furthermore, we used a stochastic kinetic Monte-Carlo simulation to predict the collective image of many sensors and how on and off rate constants affect the spatial and temporal resolution of this approach. This technique was used to image hot spots and directionality of dopamine release from neurons and other cells. We observed localized, unlabeled release sites that correlate with membrane curvature and cell morphology. We furthermore tailored SWCNTs to bind cell surface receptors such as integrins to monitor and investigate cell adhesion and migration on surfaces. These results illustrate how to use fluorescent nanosensors for quantitative chemical imaging of complex biological processes.

Versatile multiplexed super-resolution imaging of nanostructures by Quencher-Exchange-PAINT

The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps.

A Target-Triggered DNAzyme Motor Enabling Homogeneous, Amplified Detection of Proteins.

We report here the concept of a self-powered, target-triggered DNA motor constructed by engineering a DNAzyme to adapt into binding-induced DNA assembly. An affinity ligand was attached to the DNAzyme motor via a DNA spacer, and a second affinity ligand was conjugated to the gold nanoparticle (AuNP) that was also decorated with hundreds of substrate strands serving as a high-density, three-dimensional track for the DNAzyme motor. Binding of a target molecule to the two ligands induced hybridization between the DNAzyme and its substrate on the AuNP, which are otherwise unable to spontaneously hybridize. The hybridization of DNAzyme with the substrate initiates the cleavage of the substrate and the autonomous movement of the DNAzyme along the AuNP. Each moving step restores the fluorescence of a dye molecule, enabling monitoring of the operation of the DNAzyme motor in real time. A simple addition or depletion of the cofactor Mg2+ allows for fine control of the DNAzyme motor. The motor can translate a single binding event into cleavage of hundreds of substrates, enabling amplified detection of proteins at room temperature without the need for separation.

Properties of STAT1 and IRF1 enhancers and the influence of SNPs

BackgroundSTAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown.ResultsWe show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo.ConclusionsThese data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.

Toward Direct Observation of the DNA Binding Dynamics of Monomeric Type IIP Restriction Endonucleases

Restriction endonucleases (REases) are enzymes that cleave duplex DNA at a recognition site specified by a particular nucleotide sequence. These enzymes are found in a wide range of bacterial species, and serve as a defense against infection by phage. Type IIP REases are typically antiparallel homodimers that recognize a palindromic DNA sequence and cleave within or close to the recognition site. However, some Type IIP REases cleave pseudopalindromic sequences, and some of these may be active as monomers. It is hypothesized that these monomeric REases sequentially cleave the two complementary strands of duplex DNA during a single DNA binding event. Since the two strands of DNA are antiparallel, after cleaving one strand, a monomeric REase would be required to rotate about the axis perpendicular to the DNA to be properly oriented to cleave the second strand. This type of reorientation is known a “flipping”. Although only a small number of proteins have been observed reorienting in this way, protein flipping may play an important role in binding to a specific DNA sequence. Direct observation of flipping during DNA cleavage will greatly improve our understanding of how monomeric REases mediate double strand breaks. We are currently using total internal reflection fluorescence (TIRF) microscopy to observe REase-mediated cleavage of quantum dot-labeled DNA at the single-molecule level. In our highly multiplexed assay, the disappearance of a quantum dot indicates cleavage of the DNA that tethers it to a functionalized glass coverslip. We plan to utilize single-molecule Forster resonance energy transfer (smFRET) to trace individual monomeric REases in real time through the entire process of DNA cleavage from substrate binding to product release. Our observations should provide significant insight into the mechanism by which these enzymes cleave duplex DNA.

Ultrasensitive Label-Free Nanosensing and High-Speed Tracking of Single Proteins.

Label-free detection, analysis, and rapid tracking of nanoparticles is crucial for future ultrasensitive sensing applications, ranging from understanding of biological interactions to the study of size-dependent classical-quantum transitions. Yet optical techniques to distinguish nanoparticles directly among their background remain challenging. Here we present amplified interferometric scattering microscopy (a-iSCAT) as a new all-optical method capable of detecting individual nanoparticles as small as 15 kDa proteins that is equivalent to half a GFP. By balancing scattering and reflection amplitudes the interference contrast of the nanoparticle signal is amplified 1 to 2 orders of magnitude. Beyond high sensitivity, a-iSCAT allows high-speed image acquisition exceeding several hundreds of frames-per-second. We showcase the performance of our approach by detecting single Streptavidin binding events and by tracking single Ferritin proteins at 400 frames-per-second with 12 nm localization precision over seconds. Moreover, due to its extremely simple experimental realization, this advancement finally enables a cheap and routine implementation of label-free all-optical single nanoparticle detection platforms with sensitivity operating at the single protein level.

Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy.

Here we present our standard protocol for studying the binding of kinetochore proteins to microtubules as a paradigm for designing single-molecule total internal reflection fluorescence (TIRF) microscopy experiments. Several aspects of this protocol require empirical optimization, including the method for anchoring the polymer or substrate to the coverslip, the type and amount of blocking protein to prevent nonspecific protein adsorption to the glass, the appropriate protein concentration, the laser power, and the duration of imaging. Our method uses bovine serum albumin and κ-casein as blocking agents to coat any imperfections in the coverslip silanization and thereby prevent protein adsorption to the coverslip. Protein concentration and duration of imaging must be optimized for each experiment and protein of interest. Ideally, a range is determined that allows for resolution of single complexes binding to microtubules to ensure proper measurement of kinetic off rates and diffusion along microtubules. Excessively high concentrations may lead to overlapping binding of proteins on microtubules, making it impossible to resolve single binding events. The duration of imaging must be long enough to capture very low off rates (long residence time on microtubules) and we typically image at 10 frames/sec for 200 sec. The laser power can be adjusted to prevent photobleaching, but must be high enough to achieve a sufficient signal/noise ratio.