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The strategy of integrating conformational isomerization donors and chiral acceptors in a single molecule was proposed to construct white circularly polarized luminescence (WCPL) materials in this work. Consequently, a pair of dual‐emission enantiomers, namely (R/S)‐DO‐PTZ, were designed and synthesized, which displayed white emission with blue and yellow dual‐emission bands in solution and solid films with Commission Internationale de l'Eclairage (CIE) coordinates of (0.30, 0.33) and (0.33, 0.35), respectively. Meanwhile, (R/S)‐DO‐PTZ exhibited a high PLQY of up to 67 % in doped films and clear mirror‐image WCPL signals with a |glum| value of 3.0×10−3. Moreover, white circularly polarized electroluminescence (WCPEL) based on organic light‐emitting diodes (OLEDs) with (R/S)‐DO‐PTZ as emitters were also achieved with CIE coordinates of (0.32, 0.37) and EQEmax of 4.7 %, representing the state‐of‐the‐art level of white OLEDs based on single‐molecule purely organic emitters. By optimizing the device structure, warm WCPEL devices were further obtained with a |gEL| value of 2.8×10−3, CIE coordinates of (0.37, 0.48) and EQEmax of up to 15.6 %. To our knowledge, this is the first report of CP‐WOLEDs based on single‐molecule purely organic emitters. [ABSTRACT FROM AUTHOR]

[Display omitted] • Explores different approaches for expressing, purifying, and labeling GPCRs both in vitro and in live cell environments. • Discusses specific sample preparation methods for single-molecule fluorescence microscopy. • Examines the unique challenges involved in each stage of sample preparation for single-molecule GPCR experiments. • Highlights recent applications of single-molecule TIRF microscopy in investigating different classes of human GPCRs. G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography. [ABSTRACT FROM AUTHOR]

In the pursuit of sustainable technology, the synthesis of a novel biopolymer, C‐pBPV, through the polymerization of carboxymethyl cellulose sodium salt (CMC‐Na) and viologen (BPV), with ammonium persulfate as an initiator is presented. This groundbreaking process not only promises an eco‐friendly approach but also opens avenues for advanced applications in electrochromic devices (ECDs), particularly in smart windows, displays, and beyond. Departing from conventional multi‐layered approaches, the study underscores the significance of a single‐layer material. C‐pBPV serves both as an electro‐chrome and an electrolyte, making it a compelling material for ECDs. It is used to create a gel‐based electrochromic device G1 with exceptional bistable properties. Following, a systematically controllable device, G2 is accomplished, to enhance color‐switching capabilities by adding vitamin C as an electron mediator. Both devices exhibited outstanding electrochromic properties, showcasing dual‐band absorption in the Vis‐NIR region, with color transitions from transparency to a striking purple state at low voltages. Embracing an all‐in‐one layer configuration, this study set the stage for next‐generation smart windows that not only regulate solar light for energy conservation but also pioneer a more compact, user‐friendly design, prioritizing visual comfort, sustainability, and a tailored user experience. [ABSTRACT FROM AUTHOR]

Miniaturization has improved significantly in the recent decade, which has enabled the development of numerous microfluidic systems. Microfluidic technologies have shown great potential for separating desired cells from heterogeneous samples, as they offer benefits such as low sample consumption, easy operation, and high separation accuracy. Microfluidic cell separation approaches can be classified into physical (label-free) and biological (labeled) methods based on their working principles. Each method has remarkable and feasible benefits for the purposes of cancer detection and therapy, as well as the challenges that we have discussed in this article. In this review, we present the recent advances in microfluidic cell sorting techniques that incorporate both physical and biological aspects, with an emphasis on the methods by which the cells are separated. We first introduce and discuss the biological cell sorting techniques, followed by the physical cell sorting techniques. Additionally, we explore the role of microfluidics in drug screening, drug delivery, and lab-on-chip (LOC) therapy. In addition, we discuss the challenges and future prospects of integrated microfluidics for cell sorting. • Reviewing nano/microfluidics-based tumor cell isolation techniques. • Investigating physical (label-free) CTCs isolation methods. • Surveying biological (labeled) CTCs isolation techniques. • Exploring the microfluidics-based CTCs isolation in several applications. [ABSTRACT FROM AUTHOR]

It is important and challenging to analyze nanocluster structure with atomic precision. Herein, α-hemolysin nanopore was used to identify nanoclusters at the single molecule level by providing two-dimensional (2D) dwell time–current blockage spectra and translocation event frequency which sensitively depended on their structures. Nanoclusters such as Anderson, Keggin, Dawson, and a few lacunary Dawson polyoxometalates with very similar structures, even with only a two-atom difference, could be discriminated. This nanopore device could simultaneously measure multiple nanoclusters in a mixture qualitatively and quantitatively. Furthermore, molecular dynamics (MD) simulations provided microscopic understandings of the nanocluster translocation dynamics and yielded 2D dwell time–current blockage spectra in close agreement with experiments. The nanopore platform provides a novel powerful tool for nanocluster characterization. [ABSTRACT FROM AUTHOR]

Single-molecule methods allow real-time visualization of protein:DNA interactions, enabling detailed investigation of DNA damage repair. Intermediate repair stages are revealed from unsynchronized populations using single-molecule methods, thus elucidating the dynamics and mechanisms of DNA repair. Single-molecule methods have provided novel mechanistic insights into the molecular motors and protein machines involved in eukaryotic double-strand DNA break repair. Genome integrity and maintenance are essential for the viability of all organisms. A wide variety of DNA damage types have been described, but double-strand breaks (DSBs) stand out as one of the most toxic DNA lesions. Two major pathways account for the repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Both pathways involve complex DNA transactions catalyzed by proteins that sequentially or cooperatively work to repair the damage. Single-molecule methods allow visualization of these complex transactions and characterization of the protein:DNA intermediates of DNA repair, ultimately allowing a comprehensive breakdown of the mechanisms underlying each pathway. We review current understanding of the HR and NHEJ responses to DSBs in eukaryotic cells, with a particular emphasis on recent advances through the use of single-molecule techniques. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: With the continuous development of nanotechnology, single‐molecule nanopore detection has become a popular research topic. In this review, we summarize the application of biological nanopores for metal ions detection as well as overview the function of metal ions in the ion‐mediated nanopore detection of different analytes in recent decades. According to the previous reports, biological nanopores utilize two strategies to detect metal ions. First, the specific binding sites are engineered in the nanopore to slow down the translocation rate of metal ions, resulting in the diverse specific current blockage signals. Secondly, the external molecule probes are added in the detection system to interact with metal ions, leading to the characteristic changes in the signals. At the same time, the external addition of metal ions into the nanopore detection systems enhances the sensitivity and selectivity through the changes of pore charges, the coordination with analytes or indirect detection. This review provides a summary on the role of metal ions in the application of nanopore detection technology. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: With the continuous development of nanotechnology, single‐molecule nanopore detection has become a popular research topic. In this review, we summarize the application of biological nanopores for metal ions detection as well as overview the function of metal ions in the ion‐mediated nanopore detection of different analytes in recent decades. According to the previous reports, biological nanopores utilize two strategies to detect metal ions. First, the specific binding sites are engineered in the nanopore to slow down the translocation rate of metal ions, resulting in the diverse specific current blockage signals. Secondly, the external molecule probes are added in the detection system to interact with metal ions, leading to the characteristic changes in the signals. At the same time, the external addition of metal ions into the nanopore detection systems enhances the sensitivity and selectivity through the changes of pore charges, the coordination with analytes or indirect detection. This review provides a summary on the role of metal ions in the application of nanopore detection technology. [ABSTRACT FROM AUTHOR]

The hepatitis C virus (HCV) core protein (HCVcp) is the most highly conserved protein encoded by the HCV genome, and its N-terminal domain (NTDHCVcp) plays a crucial role in nucleocapsid assembly. Together, these two features make it an attractive target for antiviral therapeutics. However, NTDHCVcp is intrinsically disordered, leading to a high degree of conformational heterogeneity, and given its essential role in nucleocapsid assembly, it also tends to oligomerize at high concentrations, both of which make it difficult to characterize heterotypic intermolecular interactions between monomeric NTDHCVcp and potential therapeutics using conventional structurebased approaches. Here, we use single-molecule FRET spectroscopy to overcome these challenges and study the structural and energetic aspects of binding interactions involving different viral genotypes of NTDHCVcp and antiviral therapeutics based on antibodies, aptamers, peptides, and small molecules. Our findings highlight distinct binding mechanisms associated with these molecular interactions. For example, binding of high-affinity antibodies does not perturb the end-to-end distance of NTDHCVcp and is weakly impeded by electrostatic repulsions between similarly charged residues. Conversely, the low-nanomolar equilibrium dissociation constants of antiviral DNA aptamers arise from strong attractive electrostatic interactions that greatly decrease the end-to-end distance of NTDHCVcp. Furthermore, low-affinity antiviral peptides promote oligomerization of NTDHCVcp. Finally, the small-molecule antiviral compound we studied does not appear to affect any of our experimental observables, suggesting that binding may not alter the conformational properties of NTDHCVcp. IMPORTANCE The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals. [ABSTRACT FROM AUTHOR]

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.

Detecting conformational transitions in molecular systems is key to understanding biological processes. Here, we investigate the force variance in single-molecule pulling experiments as an indicator of molecular folding transitions. We consider cases where Brownian force fluctuations are large, masking the force rips and jumps characteristics of conformational transitions. We compare unfolding and folding data for DNA hairpin systems of loop sizes 4, 8, and 20 and the 110-amino acid protein barnase, finding conditions that facilitate the detection of folding events at low forces where the signal-to-noise ratio is low. In particular, we discuss the role of temperature as a useful parameter to improve the detection of folding transitions in entropically driven processes where folding forces are temperature independent. The force variance approach might be extended to detect the elusive intermediate states in RNA and protein folding.

Detecting conformational transitions in molecular systems is key to understanding biological processes. Here, we investigate the force variance in single-molecule pulling experiments as an indicator of molecular folding transitions. We consider cases where Brownian force fluctuations are large, masking the force rips and jumps characteristics of conformational transitions. We compare unfolding and folding data for DNA hairpin systems of loop sizes 4, 8, and 20 and the 110-amino acid protein barnase, finding conditions that facilitate the detection of folding events at low forces where the signal-to-noise ratio is low. In particular, we discuss the role of temperature as a useful parameter to improve the detection of folding transitions in entropically driven processes where folding forces are temperature independent. The force variance approach might be extended to detect the elusive intermediate states in RNA and protein folding. [ABSTRACT FROM AUTHOR]

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs. • Nsp13 unwinds dsDNA under conditions of Ca2+ with physiological relevance in cytosolic DMVs. • In single-molecule assay, unwinding activity under Ca2+ is ∼7–10 times slower than that of Mg2+. • The Ca2+-dependent unwinding activity of nsp13 driven by ATP hydrolysis. [ABSTRACT FROM AUTHOR]

We investigate through ab-initio simulations the gold-8PyrroleDiThiol-gold (Au-8PyDT) molecular quantum dot as an amperometric single-molecule sensor for the aflatoxin B1 (AFB1) detection. We study the chemical-physical interaction of AFB1 with the Au-8PyDT, and we analyze the transport characteristics of the four most probable adsorption configurations. We also investigate the link between transport modulation and adsorption mechanism. Interestingly, the investigated sensor exhibits conduction features similar to other types of quantum dot-based sensors. A significant current suppression occurs in all cases, with almost two orders of magnitude of current decrease in presence of AFB1 at 0.6 V. Considering the sensor robustness w.r.t. the specific adsorption configurations, our study motivates future research in this field. [ABSTRACT FROM AUTHOR]

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility.

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands. [ABSTRACT FROM AUTHOR]

Integrin-mediated adhesion is essential for metazoan life. Integrin binding to ligand requires an activation step prior to binding ligand that depends on direct binding of talin and kindlin to the β-integrin cytoplasmic tail and the transmission of force from the actomyosin via talin to the integrin-ligand bonds. However, the affinity of talin for integrin tails is low. It is therefore still unclear how such low-affinity bonds are reinforced to transmit forces up to 10 to 40 pN. In this study, we use single-molecule force spectroscopy by optical tweezers to investigate the mechanical stability of the talin⋅integrin bond in the presence and absence of kindlin. While talin and integrin alone form a weak and highly dynamic slip bond, the addition of kindlin-2 induces a force-independent, ideal talin⋅integrin bond, which relies on the steric proximity of and the intervening amino acid sequences between the talin- and kindlin-binding sites in the β-integrin tail. Our findings show how kindlin cooperates with talin to enable transmission of high forces required to stabilize cell adhesion. [ABSTRACT FROM AUTHOR]

Logic gates are fundamental structural components in all modern digital electronic devices. Here, nonequilibrium Green's functions are incorporated with the density functional theory to verify the thermal spin transport features of the single-molecule spintronic devices constructed by a single molecule in series or parallel connected with graphene nanoribbons electrodes. Our calculations demonstrate that the electric field can manipulate the spin-polarized current. Then, a complete set of thermal spin molecular logic gates are proposed, including AND, OR, and NOT gates. The mentioned logic gates enable different designs of complex thermal spin molecular logic functions and facilitate the electric field control of thermal spin molecular devices. [ABSTRACT FROM AUTHOR]

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single‐molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly‐expressed labeled protein, or on the addition of amyloid stains that are not protein‐specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast‐flow microfluidics and single‐molecule confocal microscopy to specifically detect α‐synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α‐synuclein aggregates down to picomolar concentrations in biologically relevant samples. [ABSTRACT FROM AUTHOR]

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer’s and Parkinson’s disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques.

KIF1A is a highly processive vesicle transport motor in the kinesin-3 family. Mutations in KIF1A lead to neurodegenerative diseases including hereditary spastic paraplegia. We applied optical tweezers to study the ability of KIF1A to generate and sustain force against hindering loads. We used both the three-bead assay, where force is oriented parallel to the microtubule, and the traditional single-bead assay, where force is directed along the radius of the bead, resulting in a vertical force component. The average force and attachment duration of KIF1A in the three-bead assay were substantially greater than those observed in the single-bead assay. Thus, vertical forces accelerate termination of force ramps of KIF1A. Average KIF1A termination forces were slightly lower than the kinesin-1 KIF5B, and the median attachment duration of KIF1A was >10-fold shorter than KIF5B under hindering loads. KIF1A rapidly reengages with microtubules after detachment, as observed previously. Strikingly, quantification enabled by the three-bead assay shows that reengagement largely occurs within 2 ms of detachment, indicating that KIF1A has a nearly 10-fold faster reengagement rate than KIF5B. We found that rapid microtubule reengagement is not due to KIF1A’s positively charged loop-12; however, removal of charge from this loop diminished the unloaded run length at near physiological ionic strength. Both loop-12 and the microtubule nucleotide state have modulatory effects on reengagement under load, suggesting a role for the microtubule lattice in KIF1A reengagement. Our results reveal adaptations of KIF1A that lead to a model of superengaging transport under load. [ABSTRACT FROM AUTHOR]

Silicon (Si) has crucial effects on plant development and stress resistance. Silicon transporters regulate Si absorption, transport, and distribution in plants. In this study, we identified and characterized the Si transporter gene family of moso bamboo (Phyllostachys edulis) and cloned seven putative Si transporter genes. Moso bamboo Si transporters contain conserved functional domains that mediate the accumulation of considerable amounts of Si. The analysis of gene duplication patterns and divergence times suggested that the expansion of the moso bamboo Si transporter family was mainly due to segmental duplications. The expression of moso bamboo Si transporter genes, which varied among organs, was significantly modulated by Si treatments. The subcellular localization analysis showed that Si transporters are plasma membrane proteins. The Si content increased in transgenic Arabidopsis overexpressing PeLsi1-1 or PeLsi1-2 , which affected vegetative and reproductive growth. Our single-particle tracking analysis revealed the four diffusion modes of PeLsi1-1 on the plasma membrane. Moreover, the particle velocity, dwell time, and motion range of PeLsi1-1 decreased in response to Si treatments. The results of this study will further clarify the molecular mechanisms underlying Si absorption and accumulation in bamboo plants. • Moso bamboo has the molecular basis of high silicon accumulation. • The excessive accumulation of silicon affected the growth of transgenic Arabidopsis. • The distribution and dynamic changes of PeLsi1-1 were affected by silicon. • A hypothetical model for silicon transport in moso bamboo seedlings was established. [ABSTRACT FROM AUTHOR]

Protein amyloid aggregation has been associated with more than 50 human disorders, including the most common neurodegenerative disorders Alzheimer's and Parkinson's disease. Interfering with this process is considered as a promising therapeutic strategy for these diseases. Our understanding of the process of amyloid aggregation and its role in disease has typically been limited by the use of ensemble-based biochemical and biophysical techniques, owing to the intrinsic heterogeneity and complexity of the process. Single-molecule techniques, and particularly diffusion-based single-molecule fluorescence approaches, have been instrumental to obtain meaningful information on the dynamic nature of the fibril-forming process, as well as the characterisation of the heterogeneity of the amyloid aggregates and the understanding of the molecular basis of inhibition of a number of molecules with therapeutic interest. In this article, we reviewed some recent contributions on the characterisation of the amyloid aggregation process, the identification of distinct structural groups of aggregates in homotypic or heterotypic aggregation, as well as on the study of the interaction of amyloid aggregates with other molecules, allowing the estimation of the binding sites, affinities, and avidities as examples of the type of relevant information we can obtain about these processes using these techniques. [ABSTRACT FROM AUTHOR]

The emerging ligation-free three-dimensional (3D) genome mapping technologies can identify multiplex chromatin interactions with single-molecule precision. These technologies not only offer new insight into high-dimensional chromatin organization and gene regulation, but also introduce new challenges in data visualization and analysis. To overcome these challenges, we developed MCIBox, a toolkit for multi-way chromatin interaction (MCI) analysis, including a visualization tool and a platform for identifying micro-domains with clustered single-molecule chromatin complexes. MCIBox is based on various clustering algorithms integrated with dimensionality reduction methods that can display multiplex chromatin interactions at single-molecule level, allowing users to explore chromatin extrusion patterns and super-enhancers regulation modes in transcription, and to identify single-molecule chromatin complexes that are clustered into micro-domains. Furthermore, MCIBox incorporates a two-dimensional kernel density estimation algorithm to identify micro-domains boundaries automatically. These micro-domains were stratified with distinctive signatures of transcription activity and contained different cell-cycle-associated genes. Taken together, MCIBox represents an invaluable tool for the study of multiple chromatin interactions and inaugurates a previously unappreciated view of 3D genome structure. [ABSTRACT FROM AUTHOR]

A major pathogenic hallmark of Alzheimer's disease is the presence of neurotoxic plaques composed of amyloid beta (Aβ) peptides in patients' brains. The pathway of plaque formation remains elusive, though some clues appear to lie in the dominant presence of Aβ1-42 in these plaques despite Aβ1-40 making up approximately 90% of the Aβ pool. We hypothesize that this asymmetry is driven by the hydrophobicity of the two extra amino acids that are incorporated in Aβ1-42. To investigate this hypothesis at the level of single molecules, we have developed a molecular "sticker-and-spacer lattice model" of unfolded Aβ. The model protein has a single sticker that may reversibly dimerise and elongate into semi-flexible linear chains. The growth is hampered by excluded-volume interactions that are encoded by the hydrophilic spacers but are rendered cooperative by the attractive interactions of hydrophobic spacers. For sufficiently strong hydrophobicity, the chains undergo liquid-liquid phase-separation (LLPS) into condensates that facilitate the nucleation of fibers. We find that a small fraction of Aβ1-40 in a mixture of Aβ1-40 and Aβ1-42 shifts the critical concentration for LLPS to lower values. This study provides theoretical support for the hypothesis that LLPS condensates act as a precursor for aggregation and provides an explanation for the Aβ1-42- enrichment of aggregates in terms of hydrophobic interactions. [ABSTRACT FROM AUTHOR]

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

RNA polymerases must initiate and pause within a complex chromatin environment, surrounded by nucleosomes and other transcriptional machinery. This environment creates a spatial arrangement along individual chromatin fibers ripe for both competition and coordination, yet these relationships remain largely unknown owing to the inherent limitations of traditional structural and sequencing methodologies. To address this, we employed long-read chromatin fiber sequencing (Fiber-seq) in Drosophila to visualize RNA polymerase (Pol) within its native chromatin context with single-molecule precision along up to 30 kb fibers. We demonstrate that Fiber-seq enables the identification of individual Pol II, nucleosome, and transcription factor footprints, revealing Pol II pausing-driven destabilization of downstream nucleosomes. Furthermore, we demonstrate pervasive direct distance-dependent transcriptional coupling between nearby Pol II genes, Pol III genes, and transcribed enhancers, modulated by local chromatin architecture. Overall, transcription initiation reshapes surrounding nucleosome architecture and couples nearby transcriptional machinery along individual chromatin fibers. [Display omitted] • Fiber-seq and FiberHMM visualize the chromatin-bound proteome on 15–20 kb fibers • Promoter-proximal pausing destabilizes and shifts downstream nucleosomes • Transcription initiation is coupled between proximal promoter and enhancer elements • Clustered tRNA and 5S rRNA genes exhibit coupled RNA Pol III transcription Tullius et al. use Fiber-seq and FiberHMM, a single-molecule, long-read genomic footprinting approach, to characterize the interplay between transcription and the broader chromatin landscape in Drosophila. They find that polymerase pausing destabilizes downstream nucleosomes and that transcription is coupled in a distance-dependent manner between nearby promoter and enhancer-promoter pairs. [ABSTRACT FROM AUTHOR]

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm–10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Commercial super-resolution (SR) imaging systems require a high budget, while current more affordable open source microscopy systems lack modularity and sometimes are too complex or lack reliability. We present miEye – a cost-effective microscope designed for high-resolution wide-field fluorescence imaging. The build is constructed using a CNC milled aluminum microscope body and commercially available optomechanics, with open-source Python-based microscope control, data visualization, and analysis software integration. The data acquisition software works robustly with commonly used industrial-grade complementary metal oxide semiconductor (iCMOS) cameras, performs IR beam back-reflection-based automatic focus stabilization, and allows for laser control via an Arduino-based laser relay. The open-source nature of the design is aimed to facilitate adaptation by the community. The build can be constructed for a cost of roughly 50 k €. It contains SM-fiber and MM-fiber excitation paths that are easy to interchange and an adaptable emission path. Also, it ensures

A major pathogenic hallmark of Alzheimer's disease is the presence of neurotoxic plaques composed of amyloid beta (Aβ) peptides in patients' brains. The pathway of plaque formation remains elusive, though some clues appear to lie in the dominant presence of Aβ1 − 42 in these plaques despite Aβ1−40 making up approximately 90% of the Aβ pool. We hypothesize that this asymmetry is driven by the hydrophobicity of the two extra amino acids that are incorporated in Aβ1−42. To investigate this hypothesis at the level of single molecules, we have developed a molecular “sticker-and-spacer lattice model” of unfolded Aβ. The model protein has a single sticker that may reversibly dimerise and elongate into semi-flexible linear chains. The growth is hampered by excluded-volume interactions that are encoded by the hydrophilic spacers but are rendered cooperative by the attractive interactions of hydrophobic spacers. For sufficiently strong hydrophobicity, the chains undergo liquid-liquid phase-separation (LLPS) into condensates that facilitate the nucleation of fibers. We find that a small fraction of Aβ1−40 in a mixture of Aβ1−40 and Aβ1−42 shifts the critical concentration for LLPS to lower values. This study provides theoretical support for the hypothesis that LLPS condensates act as a precursor for aggregation and provides an explanation for the Aβ1−42-enrichment of aggregates in terms of hydrophobic interactions.

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid–protein interactions, and they have found utility among bio-inspired technologies, including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under-explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering, and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol, and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of the vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

Fluorescent protein–DNA-binding peptides or proteins (FP-DBP) are a powerful means to stain and visualize large DNA molecules on a fluorescence microscope. Here, we constructed 21 kinds of FP-DBPs using various colors of fluorescent proteins and two DNA-binding motifs. From the database of fluorescent proteins (FPbase.org), we chose bright FPs, such as RRvT, tdTomato, mNeonGreen, mClover3, YPet, and mScarlet, which are four to eight times brighter than original wild-type GFP. Additionally, we chose other FPs, such as mOrange2, Emerald, mTurquoise2, mStrawberry, and mCherry, for variations in emitting wavelengths. For DNA-binding motifs, we used HMG (high mobility group) as an 11-mer peptide or a 36 kDa tTALE (truncated transcription activator-like effector). Using 21 FP-DBPs, we attempted to stain DNA molecules and then analyzed fluorescence intensities. Most FP-DBPs successfully visualized DNA molecules. Even with the same DNA-binding motif, the order of FP and DBP affected DNA staining in terms of brightness and DNA stretching. The DNA staining pattern by FP-DBPs was also affected by the FP types. The data from 21 FP-DBPs provided a guideline to develop novel DNA-binding fluorescent proteins. [ABSTRACT FROM AUTHOR]

Aptamers as the recognition element of the stochastic nanopore sensors have been under intense investigation. This paper reviews recent research advances in aptamer‐based nanopore sensing techniques, including the classification and selection of nanopores (biological nanopores, solid‐state nanopores, and nanopipettes), different strategies of aptamer‐based nanopore sensing, and their values and outlook for applications in areas such as environmental analysis, precision diagnosis, pharmaceutical industry, and security. Furthermore, the single‐molecule nanopore sensors have been applied to reveal the aptamer‐target interactions, such as recognition orientation, binding sites, and conformational heterogeneity dissociation kinetics at the single‐molecule level. In this review, recent research efforts to develop aptamer‐based single‐molecule nanopore sensors with high selectivity and sensitivity are highlighted, and some perspectives are drawn. [ABSTRACT FROM AUTHOR]

Acidic catecholamine metabolites, which could serve as diagnostic markers for many diseases, demonstrate an importance of accurate sensing. However, they share a highly similar chemical structure, which is a challenge in the design of sensing strategies. A nanopore may be engineered to sense these metabolites in a single molecule manner. To achieve this, a recently developed programmable nano‐reactor for stochastic sensing (PNRSS) technique adapted with a phenylboronic acid (PBA) adaptor was applied. Three acidic catecholamine metabolites, including 3,4‐dihydroxyphenylacetic acid (DOPAC), 3,4‐dihydroxymandelic acid (DHMA) and 3‐methoxy‐4‐hydroxymandetic acid (VMA) were investigated by PNRSS. Specifically, DHMA, which contains an α‐hydroxycarboxylate moiety and an adjacent cis‐hydroxyl groups on its benzene ring, reports two binding modes simultaneously resolvable by PNRSS. Assisted with the high resolution of PNRSS, direct regulation of these two binding modes by pH can also be observed. A custom machine learning algorithm was also developed to achieve automatic event classification. [ABSTRACT FROM AUTHOR]

Like helicases, CRISPR proteins such as Cas9 and Cas12a unwind DNA, but unlike helicases, these CRISPR proteins do not use ATP. Instead, they use binding energy to melt DNA locally and then utilize basepairing between guide (g) RNA and target strand to continue to unwind the DNA. CRISPR Cas9 is the most widely used tool for genome editing applications. The Cas9 endonuclease forms a complex with gRNA that can be programmed to bind a specific 20 bp segment of DNA, the protospacer. If there is enough of a sequence match between sgRNA and protospacer, Cas9 undergoes a conformational change, which activates the two nuclease domains, causing a double strand break in the DNA. We can use single-molecule FRET (smFRET) to probe the state of DNA unwinding as a function of mismatches between sgRNA and DNA. This approach can also be used to probe the position of Cas9′s HNH domain before and after cleavage. [ABSTRACT FROM AUTHOR]

The tethered molecule exhibits characteristics of both free and fixed states, with the electrodynamics involved in its diffusion, electrophoresis, and stretching processes still not fully understood. We developed a Single-Molecule Manipulation, Identification, and Length Examination (SMILE) system by integrating piezoelectric devices with nanopipettes. This system enabled successful capture and stretching of tethered double-stranded DNA within the nanopore. Our research unveiled distinct capture ( r capture ) and stretch radii ( r stretch ) surrounding the DNA's anchor point. Notably, consistent ratios of capture radius for DNA of varying lengths (2k, 4k, and 6k base pairs) were observed across different capturing voltages, approximately 1:1.4:1.83, showing a resemblance to their gyration radius ratios. However, the ratios of stretch radius are consistent to their contour length ( L 0 ), with the stretching ratio ( r stretch / L 0 ) increasing from 70 to 90% as the voltage rose from 100 to 1000 mV. Additionally, through numerical simulations, we identified the origin of capture and stretch radii, determined by the entropic elasticity-induced capture barrier and the electric field-dominant escape barrier. This research introduces an innovative methodology and outlines research perspectives for a comprehensive exploration of the conformational, electrical, and diffusion characteristics of tethered molecules.

Inhibitors of heterotrimeric G proteins are being developed as therapeutic agents. Epitomizing this approach are YM-254890 (YM) and FR900359 (FR), which are efficacious in models of thrombosis, hypertension, obesity, asthma, uveal melanoma, and pain, and under investigation as an FR-antibody conjugate in uveal melanoma clinical trials. YM/FR inhibits the Gq/11/14 subfamily by interfering with GDP (guanosine diphosphate) release, but by an unknown biophysical mechanism. Here, we show that YM inhibits GDP release by stabilizing closure between the Ras-like and α-helical domains of a Gα subunit. Nucleotide-free Gα adopts an ensemble of open and closed configurations, as indicated by single-molecule Förster resonance energy transfer and molecular dynamics simulations, whereas GDP and GTPγS (guanosine 5'-O-[gamma-thio]triphosphate) stabilize distinct closed configurations. YM stabilizes closure in the presence or absence of GDP without requiring an intact interdomain interface. All three classes of mammalian Gα subunits that are insensitive to YM/FR possess homologous but degenerate YM/FR binding sites, yet can be inhibited upon transplantation of the YM/FR binding site of Gq. Novel YM/FR analogs tailored to each class of G protein will provide powerful new tools for therapeutic investigation., Competing Interests: Competing interests statement:The authors declare no competing interest.

DNA nanostructures designed to interact with bilayer membranes are of fundamental interest as they mimic biological cytoskeletons and other membrane-associated proteins for applications in synthetic biology, biosensing, and biological research. Yet, there is limited insight into how the binary interactions are influenced by steric effects produced by 3D geometries of DNA structures and membranes. This work uses a 3D DNA nanostructure with membrane anchors in four different steric environments to elucidate the interaction with membrane vesicles of varying sizes and different local bilayer morphology. It is found that interactions are significantly affected by the steric environments of the anchors -often against predicted accessibility- as well as local nanoscale morphology of bilayers rather than on the usually considered global vesicle size. Furthermore, anchor-mediated bilayer interactions are co-controlled by weak contacts with non-lipidated DNA regions, as showcased by pioneering size discrimination between 50 and 200 nm vesicles. This study extends DNA nanotechnology to controlled bilayer interactions and can facilitate the design of nanodevices for vesicle-based diagnostics, biosensing, and protocells., (© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation has been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Super-resolution imaging, especially a single-molecule localization approach, has raised a fluorophore engineering revolution chasing sparse single-molecule dark-bright blinking transforms. Yet, it is a challenge to structurally devise fluorophores manipulating the single-molecule blinking kinetics. In this pursuit, we have developed a triggering strategy by innovatively integrating the photoactivatable nitroso-caging strategy into self-blinking sulfonamide to form a nitroso-caged sulfonamide rhodamine (NOSR). Our fluorophore demonstrated controllable self-blinking events upon phototriggered caging unit release. This exceptional blink kinetics improved the super-resolution imaging integrity on microtubules compared to self-blinking analogues. With the aid of paramount single-molecule fluorescence kinetics, we successfully reconstructed the ring structure of nuclear pores and the axial morphology of mitochondrial outer membranes. We foresee that our synthetic approach of photoactivation and self-blinking would facilitate rhodamine devising for super-resolution imaging.

Single-molecule localisation microscopy (SMLM) uses software to extract super-resolved positions from microscope images of fluorescent molecules. These localisations can then be used to render super-resolution images or analysed to extract information about molecular behaviour. The GDSC SMLM software provides a set of tools for analysing SMLM data in a single cross-platform environment. The software identifies fluorescent molecules in raw microscope images and localises their positions using stages of spot detection, spot fitting and spot rejection. The resulting localisation data set can then be visualised, cropped and filtered. A suite of downstream analysis tools enable the user to perform single-particle tracking, cluster analysis and drift correction. In addition, GDSC SMLM also provides utility tools that enable modelling of EM-CCD and sCMOS cameras as well as point spread functions (PSFs) for data simulation. The software is written in Java and runs as a collection of plugins for the ImageJ software.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example of a roadblock to replication is chemical modifications in the DNA caused by exposure to ultraviolet light. Protein dynamics are thought to play a crucial role to the molecular pathways that occur in the presence of such DNA lesions, including potential damage bypass. Therefore, many assays have been developed to study these dynamics. In this review, we discuss three methods that can be used to study protein dynamics during replisome–lesion encounters in replication reactions reconstituted from purified proteins. Specifically, we focus on ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy. We discuss two key model DNA replication systems, derived from Escherichia coli and Saccharomyces cerevisiae. The main methods of choice to study replication over the last decades have involved biochemical assays that rely on ensemble averaging. While these assays do not provide a direct readout of protein dynamics, they can often be inferred. More recently, single-molecule techniques including single-molecule fluorescence microscopy have been used to visualize replisomes encountering lesions in real time. In these experiments, individual proteins can be fluorescently labeled in order to observe the dynamics of specific proteins during DNA replication. Finally, cryo-electron microscopy can provide detailed structures of individual replisome components, which allows functional data to be interpreted in a structural context. While classic cryo-electron microscopy approaches provide static information, recent developments such as time-resolved cryo-electron microscopy help to bridge the gap between static structures and dynamic single-molecule techniques by visualizing sequential steps in biochemical pathways. In combination, these techniques will be capable of visualizing DNA replication and lesion encounter dynamics in real time, whilst observing the structural changes that facilitate these dynamics.