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A new type of material, organic salts in the crystal and crystalline powder state, with both excitation wavelength dependent (Ex‐De) fluorescence and Ex‐De phosphorescence under ambient conditions have been reported. The single component triphenylsulfonium chloride displays tunable fluorescent colors ranging from light purple to sky blue and tunable afterglow colors varying from green to yellowish green, to yellow (lifetimes: 57, 141, 66 ms, afterglow lasting for 2.1, 2.0, 2.3 s). The relationship between the structure and luminescence performance of 17 sulfonium salt derivatives confirms the pivotal roles of both the anions and the electronic and steric effect of substituent factors for luminescence. Single‐crystal analysis reveals that unique ionic bonding and intermolecular interaction can promote an ordered arrangement of organic salts in a crystal state, which then can facilitate molecular aggregation for phosphorescence generation. Theoretical calculations have also confirmed this viewpoint. More importantly, these sulfonium salt derivatives can be used for advanced multimodal anti‐counterfeiting and mitochondrial imaging, respectively. This study has not only expanded the scope of Ex‐De materials but also extended their applications. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) is rapidly evolving from an analytical method into an optical microscopy. The orthogonality of the electrochemical trigger and the optical readout distinguishes it from classic microscopy and electrochemical techniques, owing to its near‐zero background, remarkable sensitivity, and absence of photobleaching and phototoxicity. In this minireview, we summarize the recent advances in ECL imaging technology, emphasizing original configurations which enable the imaging of biological entities and the improvement of the analytical properties by increasing the complexity and multiplexing of bioassays. Additionally, mapping the (electro)chemical reactivity in space provides valuable information on nanomaterials and facilitates deciphering ECL mechanisms for improving their performances in diagnostics and (electro)catalysis. Finally, we highlight the recent achievements in imaging at the ultimate limits of single molecules, single photons or single chemical reactions, and the current challenges to translate the ECL imaging advances to other fields such as material science, catalysis and biology. [ABSTRACT FROM AUTHOR]

SUMMARY: Plasma membrane (PM)‐associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2‐domain ABA‐related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane‐delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS‐tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM‐associated ABA signaling. Single‐particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1‐related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single‐molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction. [ABSTRACT FROM AUTHOR]

Optical tweezers are a means to manipulate objects with light. Optical tweezers can obtain a nanometer space, piconewton force, and a millisecond resolution; thus, they are excellently suited for studying biological processes from the single-cell to the single-molecule level. Optical tweezers can screen and manipulate tumor cells, study single molecule, and monitor cancer treatments. Thus, optical tweezers will promote the progress of cancer research and treatments.

Optical tweezers are a means to manipulate objects with light. Optical tweezers can obtain a nanometer space, piconewton force, and a millisecond resolution; thus, they are excellently suited for studying biological processes from the single-cell to the single-molecule level. Optical tweezers can screen and manipulate tumor cells, study single molecule, and monitor cancer treatments. Thus, optical tweezers will promote the progress of cancer research and treatments. [ABSTRACT FROM AUTHOR]

Due to the ability to perform the parallel measurement of multiple single chains and exert precise control over stretching force in the sub‐nanonewton regime, magnetic tweezers (MT) are a suitable tool to study the individual chain conformations and inherent behaviors of polymers. Herein, we examined the applications of MT in studying single synthetic polymers, distinct from previous reviews focusing on biological polymers such as DNAs. We first presented an overview of the technical aspects of MT, including the imaging setup, the algorithm of three‐dimensional single particle tracking, the configuration of magnets, the calibration and control of magnetic forces, the assembly of flow cells, and the polymer tethering methods. Then, we discussed selected examples highlighting the utilization of MT in studying the chain conformation, mechanical properties, ion and ligands effect, regime transition mechanism, and polymerization dynamics of single synthetic polymers. We envision that MT can serve as a powerful toolbox for delving into the structure–property correlations at the single chain level, which provides quantitative validations for building the theoretical models of synthetic polymers. In the end, we contemplated potential avenues and opportunities for future research in this domain. [ABSTRACT FROM AUTHOR]

Water is ubiquitous and indispensable on the Earth. Its importance has long been recognized on a macroscopic scale. However, it took a long time to comprehend the impact of water molecules on the microscopic level. According to the hydrophilicity of matter, the impact of water can be divided into two categories: 1) intermolecular hydrogen bonds (H-bonds) and 2) hydrophobic interactions. In recent years, atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been used to investigate the effect of water on the single-chain properties of macromolecules. Herein, we summarize recent advances in the AFM-SMFS study on the impact of water on some kinds of synthetic polymers, biomacromolecules, and molecules containing supramolecular interactions from the two aspects of H-bonds and hydrophobic interactions. It is expected that a deeper understanding of the interactions between water molecules and macromolecules will enable the design of polymer materials with specific functionalities and properties in a bottom-up way.

G‐quadruplex (G4) is a four‐stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single‐molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging‐strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single‐stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading‐strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome. [ABSTRACT FROM AUTHOR]

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes. [ABSTRACT FROM AUTHOR]

Chloroquine has been used as a potent antimalarial, anticancer drug, and prophylactic. While chloroquine is known to interact with DNA, the details of DNA–ligand interactions have remained unclear. Here we characterize chloroquine–double-stranded DNA binding with four complementary approaches, including optical tweezers, atomic force microscopy, duplex DNA melting measurements, and isothermal titration calorimetry. We show that chloroquine intercalates into double stranded DNA (dsDNA) with a KD ~ 200 µM, and this binding is entropically driven. We propose that chloroquine-induced dsDNA intercalation, which happens in the same concentration range as its observed toxic effects on cells, is responsible for the drug's cytotoxicity. [ABSTRACT FROM AUTHOR]

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as "Bio-SOSS", leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA–protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 Å, typically within 1 Å under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures. [ABSTRACT FROM AUTHOR]

Previously limited to highly symmetrical homoleptic triple‐helical complexes [Er(Lk)3]3+, where Lk are polyaromatic tridentate ligands, single‐center molecular‐based upconversion using linear optics and exploiting the excited‐state absorption mechanism (ESA) greatly benefits from the design of stable and low‐symmetrical [LkEr(hfa)3] heteroleptic adducts (hfa−=hexafluoroacetylacetonate anion). Depending on (i) the extended π‐electron delocalization, (ii) the flexibility and (iii) the heavy atom effect brought by the bound ligand Lk, the near‐infrared (801 nm) to visible green (542 nm) upconversion quantum yield measured for [LkEr(hfa)3] in solution at room temperature can be boosted by up to three orders of magnitude. [ABSTRACT FROM AUTHOR]

Single-cell multiomics techniques have been widely applied to detect the key signature of cells. These methods have achieved a single-molecule resolution and can even reveal spatial localization. These emerging methods provide insights elucidating the features of genomic, epigenomic and transcriptomic heterogeneity in individual cells. However, they have given rise to new computational challenges in data processing. Here, we describe S ingle- c ell S ingle- m olecule m ultiple O mics P ipeline (ScSmOP), a universal pipeline for barcode-indexed single-cell single-molecule multiomics data analysis. Essentially, the C language is utilized in ScSmOP to set up spaced-seed hash table-based algorithms for barcode identification according to ligation-based barcoding data and synthesis-based barcoding data, followed by data mapping and deconvolution. We demonstrate high reproducibility of data processing between ScSmOP and published pipelines in comprehensive analyses of single-cell omics data (scRNA-seq, scATAC-seq, scARC-seq), single-molecule chromatin interaction data (ChIA-Drop, SPRITE, RD-SPRITE), single-cell single-molecule chromatin interaction data (scSPRITE) and spatial transcriptomic data from various cell types and species. Additionally, ScSmOP shows more rapid performance and is a versatile, efficient, easy-to-use and robust pipeline for single-cell single-molecule multiomics data analysis. [ABSTRACT FROM AUTHOR]

Inhomogeneity in single molecule electron transfer at the surface of lipid in a single vesicle has been explored by single molecule spectroscopic technique. In our study we took Di-methyl aniline (DMA), as the electron donor (D) and three different organic dyes as acceptor. These dyes are C153, C480 and C152 and they reside in different regions in the vesicle depending upon their preference of residence. For each probe, we found fluctuations in the single-molecule fluorescence decay, which are attributed to the variation in the reactivity of interfacial electron transfer. We found a non-exponential auto-correlation fluctuation of the intensity of the probe, which is ascribed to the kinetic disorder in the rate of electron transfer. We have also shown the power law distribution of the dark state (off time), which obeys the levy's statistics. We found a shift in lifetime distribution for the probe (C153) from 3.9 ns to 3.5 ns. This observed quenching is due to the dynamic electron transfer. We observed the kinetic disorderness in the electron transfer reaction for each dye. This source of fluctuation in electron transfer rate may be ascribed to the inherent fluctuation, occurring on the time scale of ~ 1.1 ms (for C153) of the vesicle, containing lipids. [ABSTRACT FROM AUTHOR]

Molecular motion provides a way for biomolecules to mix and interact in living systems. Quantifying their motion is critical to the understanding of how biomolecules perform its function. However, it has been a challenged task to spatially map the fast diffusion of unbound proteins in the heterogenous intracellular environment. Here we reported a new imaging technique named cumulative area based on single-molecule diffusivity mapping (CA-SMdM). The strategy is based on the comparison of single-molecule images between a shorter and longer exposure time. With longer exposure time, molecules will travel further, thus giving more blurred single-molecule images, hence implying its local diffusion rates. We validated our technique through measuring the fast diffusion rates (10–40 µm2/s) of fluorescent dye in glycerol-water mixture, and found the values fit well with Stokes-Einstein equation. We further showed that the spatially mapping of diffusivity in live cells is plausible through CA-SMdM, and it faithfully reported the local diffusivity heterogeneity in cytosol and nucleus. CA-SMdM provides an efficient way to mapping the local molecular motion, and therefore will have profound applications in probing the biomolecular interactions for living systems. [ABSTRACT FROM AUTHOR]

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function. [ABSTRACT FROM AUTHOR]

Eukaryotic gene expression is linked to chromatin structure and nucleosome positioning by ATP-dependent chromatin remodelers that establish and maintain nucleosome-depleted regions (NDRs) near transcription start sites. Conserved yeast RSC and ISW2 remodelers exert antagonistic effects on nucleosomes flanking NDRs, but the temporal dynamics of remodeler search, engagement, and directional nucleosome mobilization for promoter accessibility are unknown. Using optical tweezers and two-color single-particle imaging, we investigated the Brownian diffusion of RSC and ISW2 on free DNA and sparse nucleosome arrays. RSC and ISW2 rapidly scan DNA by one-dimensional hopping and sliding, respectively, with dynamic collisions between remodelers followed by recoil or apparent co-diffusion. Static nucleosomes block remodeler diffusion resulting in remodeler recoil or sequestration. Remarkably, both RSC and ISW2 use ATP hydrolysis to translocate mono-nucleosomes processively at ~30 bp/s on extended linear DNA under tension. Processivity and opposing push–pull directionalities of nucleosome translocation shown by RSC and ISW2 shape the distinctive landscape of promoter chromatin.

Abstract G‐quadruplex (G4) is a four‐stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single‐molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging‐strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single‐stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading‐strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

• A simple method for fluorescently labeling yeast histones and how to re-fold them into H2A-H2B dimers and H3/H4 tetramers. • A labeling strategy for histone sub-complexes. • A method for real-time visualization of nucleosomes on dsDNA. Recent advances in single molecule imaging have allowed the evolution of biochemical techniques that directly visualize protein-DNA interactions in real-time. These techniques rely on diffraction limited total internal reflection microscopy (TIRFM), and have significantly improved our understanding of RNA transcription, DNA replication, homologous recombination, and general DNA repair in the context of chromatin. Here we described a general single molecule TIRFM technique called DNA curtains to directly visualize how enzymes function on chromatinized DNA. The goal of this manuscript is to introduce the reader to methods to express and reconstitute nucleosomes on long stretches of DNA, and to directly visualize this process using DNA curtains with TIRFM. [ABSTRACT FROM AUTHOR]

Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID-19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico-newton and nano-meter resolutions. Here, a comprehensive overview of the classical BFP-DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP-DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand-receptor axes mediated cell mechanosensing. The contributions of BFP-DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP-DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense. [ABSTRACT FROM AUTHOR]

In this article, we provide an overview of our recent study on light-induced electron emission from single fullerene molecules and subnanometric optical control of their electron emission sites. After depositing molecules on a sharp metallic needle and applying strong electric fields at the apex of the needle, single-molecule protrusions appear on the apex. The electric fields are further enhanced at the protrusions and the enhanced fields drive electron emissions from the single molecules. Single-molecule electron sources can thereby be created. By irradiating such electron sources with light, we observed large modulations in the electron emission patterns. Our simulations revealed that the emission patterns represent molecular orbitals in a single molecule, a mystery scientists have been trying to get to the bottom of for the past 70 years. Our simulations also explain that light-induced modulation is driven by changes in the molecular orbitals that the emitted electrons pass through. In effect, we realize that there are subnanometric modulations in electron emission sites [ABSTRACT FROM AUTHOR]

Summary: The epidermal growth factor receptor (EGFR) is a poster child for the understanding of receptor behaviour, and of paramount importance to cell function and human health. Cloned almost forty years ago, the interest in EGFR's structure/function relationships remains unabated, not least because changes in oncogenic EGFR mutants are key drivers of the formation of lung and brain tumours. The structure of the assemblies formed by EGFR have been comprehensibly investigated by techniques such as high‐resolution X‐ray crystallography, NMR and all‐atom molecular dynamics (MD) simulations. However, the complexity embedded in the portfolio of EGFR states that are only possible in the physiological environment of cells has often proved refractory to cell‐free structural methods. Conversely, some key inroads made by quantitative fluorescence microscopy and super‐resolution have depended on exploiting the wealth of structures available. Here, a brief personal perspective is provided on how quantitative fluorescence microscopy and super‐resolution methods have cross‐fertilised with cell‐free‐derived EGFR structural information. I primarily discuss areas in which my research group has made a contribution to fill gaps in EGFR's cellular structural biology and towards developing new tools to investigate macromolecular assemblies in cells. [ABSTRACT FROM AUTHOR]

Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Chloroquine has been used as a potent antimalarial, anticancer drug, and prophylactic. While chloroquine is known to interact with DNA, the details of DNA–ligand interactions have remained unclear. Here we characterize chloroquine–double-stranded DNA binding with four complementary approaches, including optical tweezers, atomic force microscopy, duplex DNA melting measurements, and isothermal titration calorimetry. We show that chloroquine intercalates into double stranded DNA (dsDNA) with a KD ~ 200 µM, and this binding is entropically driven. We propose that chloroquine-induced dsDNA intercalation, which happens in the same concentration range as its observed toxic effects on cells, is responsible for the drug’s cytotoxicity.

Abstract Mechanical forces play a vital role in biological processes at molecular and cellular levels, significantly impacting various diseases such as cancer, cardiovascular disease, and COVID‐19. Recent advancements in dynamic force spectroscopy (DFS) techniques have enabled the application and measurement of forces and displacements with high resolutions, providing crucial insights into the mechanical pathways underlying these diseases. Among DFS techniques, the biomembrane force probe (BFP) stands out for its ability to measure bond kinetics and cellular mechanosensing with pico‐newton and nano‐meter resolutions. Here, a comprehensive overview of the classical BFP‐DFS setup is presented and key advancements are emphasized, including the development of dual biomembrane force probe (dBFP) and fluorescence biomembrane force probe (fBFP). BFP‐DFS allows us to investigate dynamic bond behaviors on living cells and significantly enhances the understanding of specific ligand‐receptor axes mediated cell mechanosensing. The contributions of BFP‐DFS to the fields of cancer biology, thrombosis, and inflammation are delved into, exploring its potential to elucidate novel therapeutic discoveries. Furthermore, future BFP upgrades aimed at improving output and feasibility are anticipated, emphasizing its growing importance in the field of cell mechanobiology. Although BFP‐DFS remains a niche research modality, its impact on the expanding field of cell mechanobiology is immense.

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER. [ABSTRACT FROM AUTHOR]

Measuring molecular binding kinetics represents one of the most important tasks in molecular interaction analysis. Surface plasmon resonance (SPR) is a popular tool for analyzing molecular binding. Plasmonic scattering microscopy (PSM) is a newly developed SPR imaging technology, which detects the out‐of‐plane scattering of surface plasmons by analytes and has pushed the detection limit of label‐free SPR imaging down to a single‐protein level. In addition, PSM also allows SPR imaging with high spatiotemporal resolution, making it possible to analyze cellular response to the molecular bindings. In this Mini Review, we present PSM as a method of choice for chemical and biological imaging, introduce its theoretical mechanism, present its experimental schemes, summarize its exciting applications, and discuss its challenges as well as the promising future. [ABSTRACT FROM AUTHOR]

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones. [ABSTRACT FROM AUTHOR]

The charge transport through single‐molecule electronic devices can be controlled mechanically by changing the molecular geometrical configuration in situ, but the tunable conductance range is typically less than two orders of magnitude. Herein, we proposed a new mechanical tuning strategy to control the charge transport through the single‐molecule junctions via switching quantum interference patterns. By designing molecules with multiple anchoring groups, we switched the electron transport between the constructive quantum interference (CQI) pathway and the destructive quantum interference (DQI) pathway, and more than four orders of magnitude conductance variation can be achieved by shifting the electrodes in a range of about 0.6 nm, which is the highest conductance range ever achieved using mechanical tuning. [ABSTRACT FROM AUTHOR]

Here we report on a label‐free electrochemiluminescence (ECL) microscopy using exceptionally low concentrations of the [Ru(bpy)3]2+ luminophore. This work addresses the central point of the minimal concentration of the ECL luminophore required to image single entities. We demonstrate the possibility to record ECL images of cells and mitochondria at concentrations down to nM and pM. This is 7 orders of magnitude lower than classically‐used concentrations and corresponds to a few hundreds of luminophores diffusing around the biological entities. Yet, it produces remarkably sharp negative optical contrast ECL images, as demonstrated by structural similarity index metric analyses and supported by predictions of the ECL image covering time. Finally, we show that the reported approach is a simple, fast, and highly sensitive method, which opens new avenues for ultrasensitive ECL imaging and ECL reactivity at the single molecule level. [ABSTRACT FROM AUTHOR]

Despite intensive research in surface enhanced Raman spectroscopy (SERS), the influence mechanism of chemical effects on Raman signals remains elusive. Here, we investigate such chemical effects through tip‐enhanced Raman spectroscopy (TERS) of a single planar ZnPc molecule with varying but controlled contact environments. TERS signals are found dramatically enhanced upon making a tip–molecule point contact. A combined physico‐chemical mechanism is proposed to explain such an enhancement via the generation of a ground‐state charge‐transfer induced vertical Raman polarizability that is further enhanced by the strong vertical plasmonic field in the nanocavity. In contrast, TERS signals from ZnPc chemisorbed flatly on substrates are found strongly quenched, which is rationalized by the Raman polarizability screening effect induced by interfacial dynamic charge transfer. Our results provide deep insights into the understanding of the chemical effects in TERS/SERS enhancement and quenching. [ABSTRACT FROM AUTHOR]

Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+‐selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe‐specific CaM regulation. We demonstrate for the first time that functional (full‐length) TRPV5 interacts with CaM in the absence of Ca2+, and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C‐lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N‐lobe calcification. Moreover, we show a Ca2+‐dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM‐dependent regulation – calmodulation – of this uniquely Ca2+‐selective TRP channel TRPV5 that involves apoCaM interaction and lobe‐specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. Key points: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis.Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+‐binding lobes.We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques.Our data support a new model for CaM‐dependent regulation of TRPV5 channel activity with CaM lobe‐specific actions and demonstrates Ca2+‐dependent binding stoichiometries.This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney. [ABSTRACT FROM AUTHOR]

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation–modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments. [ABSTRACT FROM AUTHOR]

We exploit single‐molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol‐dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes. [ABSTRACT FROM AUTHOR]

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity. [ABSTRACT FROM AUTHOR]

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double‐stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding‐degradation coordination of an exonuclease. Here, using a single‐molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA–DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2‐mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4‐bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate‐limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases. Synopsis: This study provides a dynamic understanding of the exonuclease activity of EXD2 toward RNA and DNA in double‐stranded forms. The discrete stepwise RNA–DNA hybrid cleavage of EXD2 reveals both duplex unwinding and nucleotide excision as rate‐limiting steps in nucleic acid degradation. The EXD2 exonuclease preferentially degrades RNA over DNA within RNA–DNA hybrids.EXD2 unwinds four base pairs of RNA–DNA hybrids prior to excising one nucleotide at a time.Nucleotide excision and duplex unwinding are two rate‐limiting steps in EXD2‐mediated double‐stranded nucleic acid cleavage.The frequent duplex rewinding impedes EXD2 from degrading DNA in double‐stranded forms. [ABSTRACT FROM AUTHOR]

Abstract Single‐biomolecule electronic sensing techniques are of great importance in many fields, from medical diagnosis to disease surveillance. As the physiological changes of single biomolecules can be converted into measurable electrical signals, single‐molecule electronic biosensors can realize real‐time, highly sensitive, and high‐bandwidth detection of individual intra‐ or inter‐molecular interactions. These powerful single‐molecule sensing devices have demonstrated key advantages in precisely providing rare and detailed intermediate information along reaction pathways and revealing unique properties hidden in ensemble measurements. This review summarizes significant advances in single‐molecule electronic biosensors, emphasizing biomolecule recognition, interaction, and reaction dynamics at the single‐molecule level. Sensor configurations, sensing mechanisms, and representative applications are also discussed. Furthermore, a perspective on the use of photoelectric integrated systems for synchronous sensing of the electrical and optical signals of single biomolecules is provided.

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as “Bio-SOSS”, leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA–protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 Å, typically within 1 Å under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures.