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• Snapback method as a tool for studying electrochemical surface processes. • Significant reduction in snapback distance of Au break junctions in halide electrolytes. • Electrochemical potential control confirms snapback distance reduction due to halide adsorption. 'Snapback distance' refers to the rapid increase in the size of the gap formed immediately after breaking an atomic-scale metallic contact. It is a commonly observed phenomenon in Scanning Tunnelling Microscope break junction (STM-BJ) and mechanically controlled break junction (MCBJ) experiments. Here, we show that the snapback distance measured for a gold break junction in pure water was significantly reduced in an electrolyte containing halide anions. In the case of Br−, experiments under electrochemical control provided clear evidence that this reduction was caused by halide adsorption on the surface of the gold. [ABSTRACT FROM AUTHOR]

A DNA double-strand break (DSB) jeopardizes genome integrity and endangers cell viability. Actively transcribed genes are particularly detrimental if broken and need to be repressed. However, it remains elusive how fast the repression is initiated and how far it influences the neighboring genes on the chromosome. We adopt a recently developed, very fast CRISPR to generate a DSB at a specific genomic locus with precise timing, visualize transcription in live cells, and measure the RNA polymerase II (RNAPII) occupancy near the broken site. We observe that a single DSB represses the transcription of the damaged gene in minutes, which coincides with the recruitment of a damage repair protein. Transcription repression propagates bi-directionally along the chromosome from the DSB for hundreds of kilobases, and proteasome is evoked to remove RNAPII in this process. Our method builds a foundation to measure the rapid kinetic events around a single DSB and elucidate the molecular mechanism. [Display omitted] • Single double-strand break causes transcription repression of broken genes in a few minutes • Transcription repression propagates along the chromosome for hundreds of kilobases • Rapid transcription repression is not regulated by PRC1-mediated H2A K119 ubiquitination • Proteasome-mediated RNAPII removal contributes to transcription repression and propagation He et al. apply light-activated CRISPR to induce a DNA double-strand break and observe rapid transcription repression and its propagation along the damaged chromosome. They demonstrate that proteasomes remove RNAPII around the damaged site. Their method builds a foundation to measure the kinetics after DSB and elucidate the molecular mechanism. [ABSTRACT FROM AUTHOR]

Detecting diseases at the molecular level aids in early diagnosis and treatment. However, traditional immunological detection techniques, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescence, have detection sensitivities between 10−16 and 10−12 mol/L, which are inadequate for early diagnosis. Single-molecule immunoassays can reach detection sensitivities of 10−18 mol/L and can detect biomarkers that are difficult to measure using conventional detection techniques. It can confine molecules to be detected in a small spatial area and provide absolute counting of the detected signal, offering the advantage of high efficiency and accuracy. Herein, we demonstrate the principles and equipment of two single-molecule immunoassay techniques and discuss their applications. It is shown that the detection sensitivity can be improved by 2–3 orders of magnitude compared to common chemiluminescence or ELISA assays. The microarray-based single-molecule immunoassay technique can test 66 samples in 1 h, which is more efficient than conventional immunological detection techniques. In contrast, microdroplet-based single-molecule immunoassay techniques can generate 107 droplets in 10 min, which is more than 100 times faster than a single droplet generator. By comparing the two single-molecule immunoassay techniques, we highlight our personal perspectives on the current limitations of point-of-care applications and future development trends. [Display omitted] • The principles and equipment of two single-molecule immunoassay techniques are systematically reviewed. • The point-of-care application of single-molecule immunoassay is fully discussed. • We highlight our personal perspectives on the current limitations and future development trends in single-molecule immunoassay. [ABSTRACT FROM AUTHOR]

In most microorganisms, cell division is guided by the divisome, a multiprotein complex that assembles at the equator of the cell and is responsible for the synthesis of new cell wall material. FtsZ, the first protein to assemble into this complex forms protofilaments in the cytosol which are anchored to the inner side of the cytosolic membrane by the proteins ZipA and FtsA. FtsZ protofilaments generate a force that deforms the cytosolic membrane and may contribute to the constriction force that leads to the septation of the cell. It has not been studied yet how the membrane protein anchors respond to this force generated by FtsZ. Here we studied the effect of force in the FtsZ-ZipA interaction. We used SMD and obtained the distance to the transition state of key interacting amino acids and SASA of FtsZ and ZipA through the dissociation. The SMD mechanism was corroborated by ITC, and the thermodynamic parameters ΔG0, ΔH0 and ΔS0 were obtained. Finally, we used force spectroscopy by optical tweezers to determine the lifetime of the interaction and rupture probability and their dependence on force at single molecule level. We also obtained the transition state distance, and free energy of the interaction. With the gathering of structural, thermodynamic, kinetic and force parameters we conclude that interaction between FtsZ and ZipA proteins is consistence with the highly dynamic treadmilling process and at least seven ZipA molecules are required to bind to a FtsZ protofilaments to transduce a significant force. [Display omitted] • The association between FtsZ and ZipA is an entropy based interaction. • The association between FtsZ and ZipA is driven by water molecules exit from the hydrophobic pocket of ZipA. • The residence time for the FtsZ-ZipA interaction is 12.2 s. • About eight ZipA molecules are enough to endure the bending of the cytosolic membrane at the division site. [ABSTRACT FROM AUTHOR]

Abstract Investigating the adsorption behavior of coagulation proteins on interfaces will contribute to better understating blood clotting and to the development of biocompatible materials. In this work, atomic force microscopy (AFM)-based peakforce quantitative nanomechanical mapping (PF-QNM) was combined with bioinformatics tool to study the adsorption and orientation of coagulation factor VIII (FVIII) on both hydrophilic and hydrophobic interfaces by the height and mechanical measurement of single protein molecules. We found that interfacial hydrophilicity/hydrophobicity greatly influence the heights and Young's modulus of individual proteins. Compared to on the hydrophilic mica surface, FVIII proteins appear bigger vertical sizes while similar lateral sizes on the HOPG surface. The water accessible surface area analysis indicate stronger apolar properties C1 and C2 domains than others, suggesting a preferred orientation through the strong hydrophobic interactions between HOPG and the hydrophobic residues interface of the protein domains. These results provide novel insights on the adsorption and binding mechanism of the FVIII on cell membrane and will be helpful for the design of anticoagulant materials. Graphical abstract Unlabelled Image Highlights • The height and Young's modulus of individual proteins were measured. • Protein properties were influenced by the interfacial hydrophilicity/hydrophobicity. • Human coagulation factor VIII has a preferred orientation on the hydrophobic surface. • Quantitative nanomechanical mapping and bioinformatics analysis were used. [ABSTRACT FROM AUTHOR]

Abstract We review our recent studies of photochemistry and plasmon chemistry of dimethyl disulfide, (CH 3 S) 2 , molecules adsorbed on metal surfaces using a scanning tunneling microscope (STM). The STM has been used not only for the observation of surface structures at atomic spatial resolution but also for local spectroscopies. The STM combined with optical excitation by light can be employed to investigate chemical reactions of single molecules induced by photons and localized surface plasmons. This technique allows us to gain insights into reaction mechanisms at a single molecule level. The experimental procedures to examine the chemical reactions using the STM are briefly described. The mechanism for the photodissociation reaction of (CH 3 S) 2 molecules adsorbed on metal surfaces is discussed based on both the experimental results obtained with the STM and the electronic structures calculated by density functional theory. The dissociation reaction of the (CH 3 S) 2 molecule induced by the optically excited plasmon in the STM junction between a Ag tip and metal substrate is also described. The reaction mechanism and pathway of this plasmon-induced chemical reaction are discussed by comparison with those proposed in plasmon chemistry. [ABSTRACT FROM AUTHOR]

[Display omitted] • The charge transfer mechanism of PBFDO polymers with different sizes and periods was studied through visualization. • The Raman and resonance Raman properties of long-chain PBFDO polymers were revealed. • The calculation of electron excitation for a periodic system was completed by the first principles program CP2K. • The charge transfer and I-V curves of PBFDO devices with different sizes were studied by the first principles program Quantum ATK. Single-molecule plasmas are widely used in spectroscopic studies and plasma devices, and the organic conjugated molecular chain of poly (benzodifurandione) (PBFDO) has excellent electrical conductivity and unique electronic structure. Therefore, an in-depth theoretical study of the spectroscopic, charge transfer and electron transport properties of PBFDO polymers and the analysis of physical mechanisms are essential. In this work, the absorption spectra of neutral and charged PBFDO polymers of different sizes and periodic systems of PBFDO polymers are studied theoretically. The charge transfer modes of the different absorption peaks are also given. The Raman and resonance Raman properties of long-chain PBFDO polymers under 514 nm laser were revealed. The electron transport properties and Current-Voltage Characteristic (I-V) Curves of PBFDO devices were also investigated. This work will provide the necessary theoretical guidance for the application of PBFDO in the field of nanoscale optoelectronics and the design of devices. [ABSTRACT FROM AUTHOR]

A multitude of different types of lesions is continuously introduced into the DNA inside our cells, and their rapid and efficient repair is fundamentally important for the maintenance of genomic stability and cellular viability. This is achieved by a number of DNA repair systems that each involve different protein factors and employ versatile strategies to target different types of DNA lesions. Intriguingly, specialized DNA repair proteins have also evolved to form non-functional complexes with their target lesions. These proteins allow the marking of innocuous lesions to render them visible for DNA repair systems and can serve to directly recruit DNA repair cascades. Moreover, they also provide links between different DNA repair mechanisms or even between DNA lesions and transcription regulation. I will focus here in particular on recent findings from single molecule analyses on the alkyltransferase-like protein ATL, which is believed to initiate nucleotide excision repair (NER) of non-native NER target lesions, and the base excision repair (BER) enzyme hOGG1, which recruits the oncogene transcription factor Myc to gene promoters under oxidative stress. • Functions of non-productive protein complexes in DNA repair. • Single molecule techniques in DNA repair studies. • Atomic force microscopy and fluorescence optical tweezers methodologies. [ABSTRACT FROM AUTHOR]

The Aβ42 aggregates with different structures and morphology was investigated through a single molecule label-free technique. To this end, the quartz nanopipettes were functionalized with polyethylene glycol. The set of Aβ42- epigallocatechin-3-gallate fibrils with length (from 85 nm to 250 nm) obtained by sonication was detected. The comparison of experimental and computed value of the amplitude of relative current blockade using a geometrical model show that for fibrils longer than 80 nm, the discriminating parameter is their diameter. Then, non-fibril oligomers obtain from Aβ42(Osaka) aggregation at different time seed was investigated. The analysis of the amplitude of relative current blockade shows that detected oligomers are smaller than 30 nm regardless the aggregation time. In addition, the wide distributions of the dwell time suggests the polymorph character of the sample. [Display omitted] • The Aβ42 aggregates size was investigated through a nanopipette. • For fibril, the discriminating parameter is their diameter as soon as the length is >80 nm. • Aβ42(Osaka) aggregates form for non-fibril oligomers smaller than 30 nm regardless the aggregation time. [ABSTRACT FROM AUTHOR]

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a “ f low-proteometric platform for analyzing protein concentration (FAP)” that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. [ABSTRACT FROM AUTHOR]

F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli ( E. coli ) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ε found in F O F 1 -ATP synthases of many bacteria. We focus on E. coli F O F 1 -ATP synthase, with insights into the regulatory mechanism of subunit ε arising from structural and biochemical studies complemented by single-molecule microscopy experiments. [ABSTRACT FROM AUTHOR]

Traditional fermented dairy foods including cottage cheese have been major components of the Buryatia diet for centuries. Buryatian cheeses have maintained not only their unique taste and flavor but also their rich natural lactic acid bacteria (LAB) content. However, relatively few studies have described their microbial communities or explored their potential to serve as LAB resources. In this study, the bacterial microbiota community of 7 traditional artisan cheeses produced by local Buryatian families was investigated using single-molecule, real-time sequencing. In addition, we compared the bacterial microbiota of the Buryatian cheese samples with data sets of cheeses from Kazakhstan and Italy. Furthermore, we isolated and preserved several LAB samples from Buryatian cheese. A total of 62 LAB strains (belonging to 6 genera and 14 species or subspecies) were isolated from 7 samples of Buryatian cheese. Full-length 16S rRNA sequencing of the microbiota revealed 145 species of 82 bacterial genera, belonging to 7 phyla. The most dominant species was Lactococcus lactis (43.89%). Data sets of cheeses from Italy and Kazakhstan were retrieved from public databases. Principal component analysis and multivariate ANOVA showed marked differences in the structure of the microbiota communities in the cheese data sets from the 3 regions. Linear discriminant analyses of the effect size identified 48 discriminant bacterial clades among the 3 groups, which might have contributed to the observed structural differences. Our results indicate that the bacterial communities of traditional artisan cheeses vary depending on geographic origin. In addition, we isolated novel and valuable LAB resources for the improvement of cottage cheese production. [ABSTRACT FROM AUTHOR]

For the past three decades, the field of quantum dot research has been expanding enormously, owing to the unique dynamics of charge carriers in these tiny crystals. This review provides an overview of charge carrier dynamics in photoactivated quantum dots. Specifically, it deals with the exciton fine structure, multiple exciton generation and the origin and control of photoluminescence intermittency in cadmium and lead chalcogenide quantum dots. These properties are discussed within the framework of spectroscopy and microscopy of single and multiple exciton states. Finally, this review touches on the photoluminescence- and multiple exciton- based applications of quantum dots, such as lasing and high efficiency solar cells. [ABSTRACT FROM AUTHOR]

Derivative perylene molecules deposited on Ag(111) and on NaCl(001) ultrathin layers have been investigated using low temperature STM and NC-AFM. When the metallic substrate is held at ambient temperature during evaporation, the molecules form characteristic trimers on the Ag(111) surface and interact through their polar groups. Close to the steps, the molecules form linear structures and seems to stand side by side. On the other hand, after deposition on a substrate cooled at liquid helium temperature, single molecules are observed both on metal and on NaCl. On the ultrathin insulator layers, the STM images present characteristic contrasts related to the molecular orbitals which favors the localization of aldehyde groups. In this case, the lateral molecular interactions may induce the formation of small assemblies in which the electronic levels are slightly shifted. A possible interpretation of this phenomenon is to take into account polar interactions and charge transfer between neighboring molecules. [ABSTRACT FROM AUTHOR]

Single Molecule Orientation and Localization Microscopy (SMOLM) is gaining an increasing interest in the community of localization microscopy, due to the capability to monitor orientational information in addition to spatial reconstruction. In many cases, molecule's orientations are not random and their linker to a protein of interest is sufficiently rigid to be able to report orientation information from this protein. While several strategies exist to report single molecule orientation based on polarization splitting and point spread function engineering, the effect of the incident polarization has been often neglected by supposing its effect embedded in the retrieved angular parameters, or by supposing the excitation to be isotropic or the rotational diffusion to be very fast. In this work we quantify the amount of possible bias brought in SMOLM readout due to the incident polarization effect, using analytical derivations. We illustrate this effect experimentally on single molecules attached to a surface in presence of wobbling. [ABSTRACT FROM AUTHOR]

As transmembrane proteolytic enzyme, matrix metalloproteinase-14 (MMP14) regulates cell migration and cancer metastasis, but how it works at the single molecule level is unclear. Molecular localization is closely related to its function, and revealing its spatial assemble details is thus helpful to understand bio-function. Here, we apply aptamer probe and dSTORM to characterize MMP14 distribution. With demonstrating labeling properties of the probe, we investigate the specific distributed pattern of MMP14 on various cell membranes with different migratory capacities, and find that MMP14 mostly aggregate in clustering state, which becomes more significant with enhancing its hydrolysis efficiency on high-migratory cells. Lots of MMP14 are revealed to be co-localized with its substrate PTK7, and this colocalization decreases with weakening cell migration, suggesting that MMP14 may coordinate cell migration by altering its spatial relationship with substrate proteins. This work will promote a deep understanding of the roles of MMP14 in cell migration and cancer metastasis. [Display omitted] • Verification of the labeling specificity and labeling advantages of aptamer probe in dSTORM imaging. • Differential distribution patterns of MMP14 on the surface of different functional membranes. • The high expression and aggregation distribution of MMP14 are specific to cancer cells. • The detailed relationship between MMP14 assembly and its biological function. • MMP14 regulates cell migration by changing the co-location of function-related proteins. [ABSTRACT FROM AUTHOR]

Circulating autoantibodies against glutamatergic N -methyl-D-aspartate receptor (NMDAR) have been reported in a proportion of patients with psychotic disorders, raising hopes for more appropriate treatment for these antibody-positive patients. However, the prevalence of circulating autoantibodies against glutamatergic NMDAR in psychotic disorders remains controversial, with detection prevalence rates and immunoglobulin classes varying considerably between studies, perhaps because of different detection methods. Here, we compared the results of serum assays for a large cohort of patients with first-episode psychosis using classical cell-based assays in three labs and a single molecule-based imaging method. Most assays and single molecule imaging in live hippocampal neurons revealed the presence of circulating autoantibodies against glutamatergic NMDAR in approximately 5% of patients with first-episode psychosis. However, some heterogeneity between cell-based assays was clearly observed, highlighting the urgent need for new sensitive methods to detect the presence of low-titer autoantibodies against glutamatergic NMDAR in seropositive patients who cannot be clinically identified from seronegative ones. [ABSTRACT FROM AUTHOR]

Summary Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. [ABSTRACT FROM AUTHOR]

This work describes how nanostructured thin films obtained from the self-assembly of block copolymers (BCPs) systems can be used as a nanoelectrode array (NEA) that can be programmed to specifically detect targeted molecules. Namely, poly(styrene-b-methacrylate) (PS-b-PMMA) thin films, after removal of PMMA phase, produced regular spaced 560 pores μm-2 with 16 nm of diameter. The nanopores were then chemically modified by the introduction of β-cyclodextrin (β-CD) molecules. By using the supramolecular interaction of β-CD and ferrocene (Fc), the pores could be programed by the introduction of molecules linked to Fc able to interact with target species. In the model system shown here, the linker had a biotin unit, aiming the detection of streptavidin. By changing the linker, other molecules can also be detected. This concept opens a window to many possibilities, including the development of devices for fast and versatile molecule detection. [ABSTRACT FROM AUTHOR]

We present a study of coherent tunneling across rigid rod-like Oligo(phenylene-ethylene) molecules coupled to gold leads for different molecular lengths and anchoring groups. We employ a Density Functional Tight Binding (DFTB) Hamiltonian combined with Green’s function formalism. We show that the correct decay β of the tunneling probability with respect to the molecular length is only recovered after a proper renormalization of the energy levels for the occupied and unoccupied molecular orbitals. To this end, we implement a density matrix based spectral adjustment. After correction a β independent of the anchoring group is obtained, in good quantitative agreement with available experiments and previous ab initio density functional calculations. [ABSTRACT FROM AUTHOR]

Single molecule assays were performed on the enzyme E. coli β-glucuronidase using a capillary electrophoresis-based protocol. Electrophoretic mobility, catalytic rate and activation energy of catalysis were all found to be heterogeneous. The average mobility at 22 °C was −1.1 × 10 −8 ± 0.1 m 2 V −1 s −1 (N = 49) with a total range of −0.6 to −1.3 × 10 −8 m 2 V −1 s −1 . The range in electrophoretic mobility suggests that the differences in shape or charge of the individual molecules underlying the heterogeneity are likely minimal. The average catalytic rate at 22 °C was 37,000 ± 19,000 min −1 (N = 49) with a total range of 14,000 to 130,000 min −1 . Both of these properties were measured simultaneously for each of the molecules. There was a weak correlation (r 2 = 0.43) between mobility and rate with the molecules with a less negative mobility having a tendency to have a higher rate. The average activation energy of catalysis, as determined by comparing rates at 22 and 35 °C, was found to be 48 ± 18 kJ mol −1 (N = 7) with a total range of 18–66 kJ mol −1 . [ABSTRACT FROM AUTHOR]

The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules. [Display omitted] • Nanoscale map of the hippocampal formation extracellular space • The extracellular space characteristics differ between areas and layers • The CA1 stratum radiatum extracellular space is smaller but more fluid than in CA3 • The extracellular space characteristics tune immunoglobulin distribution Grassi et al. describe the nanoscale organization of the extracellular space of the rodent hippocampus. Its physical properties differ strongly across the hippocampus, in particular between the stratum radiatum of CA1/3. These marked differences go hand in hand with differences in the dynamics and distribution of immunoglobulins in these areas. [ABSTRACT FROM AUTHOR]

Summary Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility. [ABSTRACT FROM AUTHOR]

Single molecule studies allow for the direct observation of polymer dynamics in dilute and concentrated solutions, thereby revealing polymer chain conformations and molecular sub-populations that may be obscured in ensemble-level measurements. Over the past two decades, researchers have used DNA as a model system to study polymer dynamics at the molecular level. The vast majority of studies have focused on linear DNA molecules; however, researchers have recently begun to study polymers with complex topologies and architectures at the single molecule level. Here, we explore recent work in single polymer dynamics focused on topologically complex DNA, including knots, ring polymers, and branched polymers. Experimental, computational, and theoretical advances have enabled in-depth studies of topologically complex DNA, with recent efforts focused on complex molecular conformations, intermolecular interactions, and topology-dependent dynamics. In this article, we highlight recent work aimed at understanding the interplay between molecular-scale behavior and the emergent properties of polymeric materials. [ABSTRACT FROM AUTHOR]

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude enhancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4) 2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes promising candidates for biological labeling and microscopy applications and single molecule tracking. [Display omitted] • Squaraine Figure Eight (SF8) dyes encapsulate the emissive fluorochrome. • SF8 dyes exhibit longer times to photobleaching. • Compared to Cy5, the dye SF8(D4) 2 permits twenty times larger photon yields. • SF8 dyes will be especially useful for photon-intensive microscopy. [ABSTRACT FROM AUTHOR]

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins. [Display omitted] • The Smc5/6 holocomplex can form oligomers and translocate on double-stranded DNA • The complex can simultaneously bind single-stranded and double-stranded DNA • Smc5/6 is recruited to ssDNA-dsDNA junction structures mimicking replication forks • The holocomplex indirectly stabilizes the ssDNA-binding protein RPA on ssDNA Tanasie et al. combine single-molecule microscopy and force manipulation to investigate the interaction of the Smc5/6 holocomplexes with fork junction structures mimicking DNA replication and repair intermediates. They show that Smc5/6 targets and stabilizes these DNA substrates, suggesting a model for the Smc5/6-mediated recognition of replication stress or DNA lesions. [ABSTRACT FROM AUTHOR]

Various techniques to measure the force-extension relationship of individual polymer chains with the atomic force microscope (AFM) are compared. Reliable stretching force profiles can be obtained with the nano-handling technique, which involves imaging of the measured individual polymer molecules prior and after the force experiment. Results originating from the classical pulling technique, which relies on dense adsorbed polymer layers, must be interpreted with care. Comparison with the nano-handling technique reveals that the stretching response of individual molecules can be obtained with the pulling technique, provided the adsorbed polymer layer is dilute. For denser adsorbed layers, such experiments may not reflect the correct stretching response. The discrepancies seem to be related to polymer–polymer interactions and entanglements within the adsorbed layer. AFM imaging of the adsorbed layers can provide an indication concerning the relevance of such entanglement effects. Similar caveats apply to fishing experiments, where polymers are adsorbed to the AFM tip and not to the substrate. [ABSTRACT FROM AUTHOR]

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca 2+ -mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks (‘oligoGs’) using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4 Ca 2+ ions, with both shorter and longer oligomers forming weaker links. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level. [ABSTRACT FROM AUTHOR]

The calcium signaling protein calmodulin (CaM) interacts with many target proteins inside the cell to regulate a wide range of biological signals. CaM's availability to propagate signals depends on its mobility, which may be regulated by interactions with multiple target proteins. We detected single molecules of CaM labeled with a fluorescent dye and injected into living HEK 293 cells, and we used high-speed, wide-field, single-molecule imaging to track single CaM molecules. Single-molecule trajectories were analyzed to characterize the motions of individual CaM molecules. Single-molecule localization resolved CaM positions with a position accuracy of < 100 nm, permitting sub-diffraction imaging of features with localized CaM that form in response to increased free Ca 2+ . Single-molecule tracking demonstrated the presence of a wide range of mobilities of individual calmodulin molecules in a cell, with diffusion coefficients ranging from < 0.01 μm 2 s −1 to ~ 5 μm 2 s −1 , whereas analysis by spatio-temporal image correlation spectroscopy revealed faster-moving components with diffusion coefficients of > 10 μm 2 s −1 . For molecules confined to small regions of the cell, super-resolved images of presumed signaling complexes were recovered. Individual trajectories were classified as normal diffusion, confined diffusion, or directed motion, and could suggest how the individual CaM molecules were bound in the cell. The results show that interactions of CaM with target proteins result in decreased translational mobilities of a significant fraction of CaM molecules inside cells. The work presented here illustrates methods that can characterize location, mobilities, and the availability of signaling molecules in live cells. [ABSTRACT FROM AUTHOR]

In this study, a microscopic probabilistic model of chromatography that establishes a conceptual link between single-molecule dynamics observation at liquid–solid interfaces and chromatographic experiments is described. This model is based on the discrete Lévy representation of stochastic processes and has the great advantage that it can be directly applied to the raw data set of single-molecule observations. The information contained in the molecular measurements includes some erratic rare events that are potentially very informative. Because experimental data need not be processed by mathematical–statistical transformation, application of this model preserves all the information that could be lost in an ensemble-averaged representation. In this approach, single-molecule experiments and stochastic interpretation are combined. It is of great importance to investigate superficial and interfacial phenomena in different areas, such as adsorption mechanisms in chromatography and mechanisms of biological activity, and to track the behavior of individual molecules in living cells. [ABSTRACT FROM AUTHOR]

Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction. [ABSTRACT FROM AUTHOR]

Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. [ABSTRACT FROM AUTHOR]

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines. [ABSTRACT FROM AUTHOR]

The bacterial enzyme EndoS specifically cleaves glycans bound to immunoglobulin G (IgG) molecules. Because this deglycosylation procedure leads to a diminished immune response, this enzyme has potential applications as a therapeutic for autoimmune disorders. Although the diminished immune response is attributed to a structural change in the Fc region of IgG antibodies, the specific nature of this structural change is not known due to the variety of results obtained by different experimental approaches. In order to better understand how EndoS deglycosylation impacts the structure of the Fc region of IgG antibodies, we have conducted single molecule Förster resonance energy transfer (FRET) studies of dye-labeled, freely diffusing antibodies. A comparison of the FRET efficiency histograms obtained for glycosylated and EndoS deglycosylated antibodies indicates that the Fc region can take on a wider variety of structures upon deglycosylation. This is demonstrated by the presence of additional peaks in the FRET efficiency histogram for the deglycosylated case. [ABSTRACT FROM AUTHOR]

Heterogeneous electron transfer (ET) of the redox protein, wild-type azurin (wt-Az) from Pseudomonas aeruginosa , was monitored at the single-molecule (SM) level by fluorescence resonance energy transfer (FRET), one electron at a time. Azurin molecules were labeled with an organic fluorophore (Cy5), and the FRET-coupling between Cy5 and the redox center (copper) was used to study ET to a semi-transparent, 10 nm thin gold electrode in an optical configuration. By using a confocal microscope and a bipotentiostat for control of the electrode potential, the oxidation and reduction processes of individual Az–Cy5 molecules were monitored. In the oxidized state of the redox center of the azurin molecule, the fluorescence emission of the covalently attached Cy5 was largely quenched by FRET (‘off’-state), whereas the emission was recovered upon reduction (‘on’-state). The work presented here, shows directly controlled single redox switching events of an individual redox protein and its thermodynamic dispersion. We show that the distribution of midpoint potentials ( E 0 ) of individual azurin molecules peaks at 45.7±0.5 mV with a full width at half maximum of 15 mV vs saturated calomel electrode (SCE). [ABSTRACT FROM AUTHOR]

We review several representative experimental results of action spectroscopy (AS) of single molecules on metal surfaces using a scanning tunneling microscope (STM) by M. Kawai’s group over last decade. The experimental procedures to observe STM-AS are described. A brief description of a low-temperature STM and experimental setup are followed by key experimental techniques of how to determine an onset bias voltage of a reaction and how to measure a current change associated with reactions and finally how to observe AS for single molecule reactions. The experimental results are presented for vibrationally mediated chemical transformation of trans -2-butene to 1.3-butadiene molecule and rotational motion of a single cis -2-butene molecule among four equivalent orientations on Pd(1 1 0). The AS obtained from the motion clearly detects more vibrational modes than inelastic electron tunneling spectroscopy with an STM. AS is demonstrated as a useful and novel single molecule vibrational spectroscopy. The AS for a lateral hopping of water dimer on Pt(1 1 1) is presented as an example of novelty. Several distinct vibrational modes are detected as the thresholds in the AS. The assignment of the vibrational modes determined from the analysis of the AS is made from a view of the adsorption geometry of hydrogen-bond donor or acceptor molecules in water dimer. A generic theory of STM-AS, i.e., a reaction rate or yield as a function of bias voltage, is presented using a single adsorbate resonance model for single molecule reactions induced by the inelastic tunneling current. Formulas for the reaction rate R ( V ) and Y ( V ) , i.e., reaction yield per electron Y ( V ) = eR ( V ) / I are derived. It provides a versatile framework to analyze any vibrationally mediated reactions of single adsorbates on metal surfaces. Numerical examples are presented to demonstrate generic features of the vibrational generation rate and Y ( V ) at different levels of approximations and to show how the effective broadening of the vibrational density of states (as described by Gaussian or Lorentzian functions) manifest themselves in Y ( V ) near the threshold bias voltage corresponding to a vibrational excitation responsible for reactions. A prefactor of Y ( V ) is explicitly derived for various types of elementary processes. Our generic formula of Y ( V ) also underlines the need to observe Y ( V ) at both bias voltage polarities, which can provide additional insight into the adsorbate projected density of states near the Fermi level within a span of the vibrational energy. The theory is applied to analysis of some highlights of the experimental results: Xe transfer, hopping of a single CO molecule on Pd(1 1 0), a dissociation of a single dimethyl disulfide (CH 3 S) 2 and a hopping of a dissociated product, i.e., single methyl thiolate CH 3 S on Cu(1 1 1). It underlines that an observation of Y ( V ) at both bias polarities permits us to certain insight into the molecular alignment with respect to the Fermi level. [ABSTRACT FROM AUTHOR]

All organisms must dramatically compact their genomes to accommodate DNA within the cell. Bacteria use a set of DNA-binding proteins with low sequence specificity called nucleoid-associated proteins (NAPs) to assist in chromosome condensation and organization. By bending or bridging DNA, NAPs also facilitate chromosome segregation and regulate gene expression. Over the past decade, emerging single-molecule and chromosome conformation capture techniques have investigated the molecular mechanisms by which NAPs remodel and organize the bacterial chromosome. In this review we describe how such approaches reveal the biochemical mechanisms of three NAPs that are believed to facilitate DNA bridging: histone-like nucleoid structuring protein (H-NS), ParB, and structural maintenance of chromosomes (SMC). These three proteins form qualitatively different DNA bridges, leading to varied effects on transcription and chromosome segregation. [ABSTRACT FROM AUTHOR]

We use a scanning tunneling microscope-based break-junction technique to compare the probability of the formation of a single-molecule junction for a series of amine-terminated oligophenyl and alkane species using either Ag or Au as electrodes. For all molecules, we find that there is a significantly higher probability of junction formation when using Ag electrodes than with Au electrodes. We also find that longer molecules have a higher probability than shorter molecules to form a junction for both Ag and Au electrodes. For all molecules, the measured molecular junction length that is formed with the Ag electrodes was longer than that formed with Au electrodes. Furthermore, we can make a single atomic oxygen junction and can measure its conductance using Ag electrodes. These observations are attributed to a narrower gap of the Ag electrodes compared to that of the Au electrodes after the metal contact ruptures. Since there is a high probability of a molecular junction forming when using Ag electrodes, we can therefore perform a statistical analysis within the context of the material properties that are suitable for future molecular electronics. [ABSTRACT FROM AUTHOR]

[Display omitted] • Exciplex emission was achieved by a single molecular system with flexible chain. • Ho-exciplex eliminates the phase separation drawback of physical blended exciplex. • Ho-exciplex can enable the fabrication of fully-solution-processed OLEDs. Exciplex has long been recognized as a mixture of donor and acceptor components. Here, for the first time, it was demonstrated that exciplex emission can also be achieved by single pure molecule, we named it as homojunction exciplex (ho-exciplex). By using the flexible alkyl chain to link the donor and acceptor unit, the result compound not only maintain efficient exciplex emission property, but also eliminate the notorious phase separation drawback. Different from the conventional heterojunction exciplex (he-exciplex), such unique molecular concept of homojunction system will provides a bright avenue toward structure-simplified and high-stable OLEDs without sophisticated stoichiometric and physical blending process. By using such a single molecular PhTRZ-CzTPA as both emitter and host, the solution-processed device achieved a comparable electroluminescence performance with its hybrid blending he-exciplex counterpart. Importantly, the unified molecule of PhTRZ-CzTPA makes it superior solvent resistance to isopropanol, which enable the fabrication of fully-solution-processed device with all spin-coated organic layers. Therefore, the homojunction strategy will open a new avenue for exciplex design and application, not only in OLEDs, but also other semiconductor devices or sensors. [ABSTRACT FROM AUTHOR]

To explore the nature of water insolubility of polyformaldehyde (POM), we utilize single-molecule atomic force microscopy (AFM) and molecular dynamics (MD) simulations to study the possible interactions between POM and water at the single-chain level. Single-molecule force-extension curves of POM obtained in nonane and deionized water showed a marked deviation in the middle force region, which is similar to that observed in polyethylene glycol (PEG). The results indicate that force-induced hydrogen bonding between POM and water occurs upon initial stretching, followed by a dehydration process upon high force stretching. MD simulations show that POM chain itself is able to form H-bonds with water, but this potential is heavily hindered by the native conformation in the crystal. This finding not only provides new insight into the understanding of water insolubility of POM, but also helps the rational design of advanced polymer materials in the future. [Display omitted] • Single-molecule F-E curves of POM obtained in nonane and water showed a marked deviation. • The deviation of the F-E curves obtained in nonane and water of POM is similar to that of PEG. • Force-induced H-bonding between POM and water should occur upon initial stretching. • The force-induced H-bonding between POM and water is supported by MD simulations. [ABSTRACT FROM AUTHOR]

Solid-state nanopore has the ability to detect proteins at a single-molecule level with its high sensitivity, high-throughput, and low cost. Improvements in fabrication, functionalization, and characterization of solid-state nanopores keep evolving. Various analytical methods targeted towards diagnostic applications using nanopore-based devices are appearing. This review article provides an overview of recent progress in the field of solid-state and biological nanopores for protein detection in a complex analyte. The advantages and challenges involved in nanopore sensing have been discussed. Further, the review surpasses the steady-state resistive pulse techniques of sensing and incorporate transient variations in the nanopore conductance. Application of the power spectral density of these fluctuations toward sensing has been highlighted with importance on reducing the detection limit in a complex environment. Lastly, the current problems and future perspectives have been discussed with a perspective to increase nanopores performance towards diagnostic applications in complex medium. [Display omitted] • Overview of recent developments of solid-state and biological nanopores for protein sensing in a complex analyte. • Functionalization strategies of both solid-state and biological nanopores. • Application of the power spectral density of transient nanopore conductance fluctuations in a complex environment. [ABSTRACT FROM AUTHOR]

We review recent advances in our ability to characterise biomolecular structure, interactions and associated dynamics by mass photometry (MP), the label-free detection and mass measurement of individual biomolecules in solution. Molecular counting and identification provides direct access to relative abundance, and thereby affinities, while associated dynamics yield on- and off-rates. The molecular resolution afforded by MP enables these measurements as a function of stoichiometry and assembly at equilibrium, as opposed to the majority of existing solution-based methods. Together with future improvements in terms of assays and technological performance, MP is likely to provide mechanistic details of complex biomolecular processes. • Mass photometry provides quantitative, label-free, detection and mass measurement of individual biomolecules. • It can be applied to a wide variety of biomolecules while operating in native solution. • Stoichiometry, energetics and dynamics can be studied in heterogeneous mixtures. [ABSTRACT FROM AUTHOR]

The folding dynamics of riboswitches are central to their ability to modulate gene expression in response to environmental cues. In most cases, a structural competition between the formation of a ligand-binding aptamer and an expression platform (or some other competing off-state) determines the regulatory outcome. Here, we review single-molecule studies of riboswitch folding and function, predominantly carried out using single-molecule FRET or optical trapping approaches. Recent results have supplied new insights into riboswitch folding energy landscapes, the mechanisms of ligand binding, the roles played by divalent ions, the applicability of hierarchical folding models, and kinetic vs. thermodynamic control schemes. We anticipate that future work, based on improved data sets and potentially combining multiple experimental techniques, will enable the development of more complete models for complex RNA folding processes. This article is part of a Special Issue entitled: Riboswitches. [ABSTRACT FROM AUTHOR]

The fate of a cell depends on its ability to repair the many double-stranded DNA breaks (DSBs) that occur during normal metabolism. Improper DSB repair may result in genomic instability, cancer, or other genetic diseases. The repair of a DSB can be initiated by the recognition and resection of a duplex DNA end to form a 3'-terminated single-stranded DNA overhang. This task is carried out by different single-strand exonucleases, endonucleases, and helicases that work in a coordinated manner. This manuscript reviews the different single-molecule approaches that have been employed to characterize the structural features of these molecular machines, as well as the intermediates and products formed during the process of DSB repair. Imaging techniques have unveiled the structural organization of complexes involved in the tethering and recognition of DSBs. In addition to that static picture, single molecule studies on the dynamics of helicase-nuclease complexes responsible for the processive resection of DSBs have provided detailed mechanistic insights into their function. [ABSTRACT FROM AUTHOR]

Despite three decades of biochemical and structural analysis of the prokaryotic nucleotide excision repair (NER) system, many intriguing questions remain with regard to how the UvrA, UvrB, and UvrC proteins detect, verify and remove a wide range of DNA lesions. Single-molecule techniques have begun to allow more detailed understanding of the kinetics and action mechanism of this complex process. This article reviews how atomic force microscopy and fluorescence microscopy have captured new glimpses of how these proteins work together to mediate NER. [ABSTRACT FROM AUTHOR]

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit - searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair. [ABSTRACT FROM AUTHOR]

Highlights: [•] Single molecule FRET can be used to study the structure of the Fc region of IgG. [•] FRET results for glycosylated IgG agree with crystal structure results. [•] FRET results for deglycosylated IgG indicate a wide range of Fc conformations. [•] Deglycosylation appears to enhance the flexibility of the Fc region of IgG. [Copyright &y& Elsevier]

Highlights: [•] Molecules involved in transcription have non-random spatial distributions in cells. [•] Spatial organization may offer a new gene regulation mechanism at the cellular level. [•] More studies are needed to correlate functionality with spatial localization patterns. [ABSTRACT FROM AUTHOR]