Optimization of hybridization chain reaction for imaging single RNA molecules in Drosophila larvae.

In situ hybridization techniques are powerful methods for exploring gene expression in a wide range of biological contexts, providing spatial information that is most often lost in traditional biochemical techniques. However, many in situ hybridization methods are costly and time-inefficient, particularly for screening-based projects that follow on from single-cell RNA sequencing data, which rely on of tens of custom-synthetized probes against each specific RNA of interest. Here we provide an optimized pipeline for Hybridization Chain Reaction (HCR)-based RNA visualization, including an open-source code for optimized probe design. Our method achieves high specificity and sensitivity with the option of multiplexing using only five pairs of probes, which greatly lowers the cost and time of the experiment. These features of our HCR protocol are particularly useful and convenient for projects involving screening several genes at medium throughput, especially as the method include an amplification step, which makes the signal readily visible at low magnification imaging.

Visualizing, quantifying and mapping chromatin remodelers at work with single-molecule and single-cell imaging.

Chromatin remodeling, carried out by four major subfamilies of ATP-dependent remodeler complexes across eukaryotes, alleviates the topological challenge posed by nucleosomes to regulate genome access. Recently, single-molecule and single-cell imaging techniques have been widely employed to probe this crucial process, both in vitro and in cellulo. Herein, we provide an integrated account of key recent efforts that leverage these approaches to visualize, quantify and map chromatin remodelers at work, elucidating diverse aspects of the remodeling process in both space and time, including molecular mechanisms of DNA wrapping/unwrapping, nucleosome translocation and histone exchange, dynamics of chromatin binding/target search and their intranuclear organization into hotspots or phase condensates, as well as functional coupling with transcription. The mechanistic insights and quantitative parameters revealed shed light on a multi-modal yet shared landscape for regulating remodeling across molecular and cellular scales, and pave the way for further interrogating the implications of its misregulation in disease contexts.

Bacterial and fungal aerosols in poultry houses: PM2.5 metagenomics via single-molecule real-time sequencing.

Microbial aerosol contamination is a common problem in poultry farms, posing potential health risks to poultry and their caretakers. Exploring the distribution and diversity of the microbial community in poultry farm aerosols is crucial for effective mitigation strategies. The composition of bacterial and fungal aerosols is poorly understood, particularly the prevalence of potential pathogenic microorganisms in fine particulate matter (PM2.5) in various types of poultry houses. In this study, 27 PM2.5 samples were collected from 5 chicken houses and 4 duck houses in Shandong Province, China. Species-level diversity of bacterial and fungal components in PM2.5 samples was determined using advanced single-molecule real-time sequencing (SMRT) technology, based on the 16S and internal transcribed spacer 1 (ITS) ribosomal genes. Microbial diversity and community composition varied significantly across the different poultry house. Notably, duck houses had higher concentrations (p < 0.01) of PM2.5 (92.8-143.1 µg/m3) than chicken houses (42.0-56.4 µg/m3). Furthermore, microbial variation in PM2.5 samples was associated with the type of poultry facility. The predominant pathogenic microorganisms included Aspergillus sydowii, Penicillium sp., Aspergillus insolitus, Cladosporium sp., Aspergillus sp., Aspergillus pseudoglaucus, Cladosporium sp. C4092-2-PD1, and Colletotrichum sp., all of which were classified as second category of pathogens. Aspergillus sydowii and Penicillium sp. were the dominant species in chicken houses, while Cladosporium sp., Aspergillus sp., and Aspergillus pseudoglaucus were the dominant species identified in duck houses. To our knowledge, this study is the first to investigate bacterial and fungal diversity in PM2.5 samples collected from various types of poultry houses. These findings advance our understanding of poultry environmental microbiology and have important implications for safeguarding the health of both poultry and their caretakers.

Conformational dynamics of SARS-CoV-2 Omicron spike trimers during fusion activation at single molecule resolution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron entry involves spike (S) glycoprotein-mediated fusion of viral and late endosomal membranes. Here, using single-molecule Förster resonance energy transfer (sm-FRET) imaging and biochemical measurements, we directly visualized conformational changes of individual spike trimers on the surface of SARS-CoV-2 Omicron pseudovirions during fusion activation. We observed that the S2 domain of the Omicron spike is a dynamic fusion machine. S2 reversibly interchanges between the pre-fusion conformation and two previously undescribed intermediate conformations. Acidic pH shifts the conformational equilibrium of S2 toward an intermediate conformation and promotes the membrane hemi-fusion reaction. Moreover, we captured conformational reversibility in the S2 domain, which suggests that spike can protect itself from pre-triggering. Furthermore, we determined that Ca2+ directly promotes the S2 conformational change from an intermediate conformation to post-fusion conformation. In the presence of a target membrane, low pH and Ca2+ stimulate the irreversible transition to S2 post-fusion state and promote membrane fusion.

Dinuclear Dysprosium Compounds: The Importance of Rigid Bridges.

We report the synthesis, structures and magnetic behaviour of two isostructural dinuclear Dy3+ complexes where the metal ions of a previously reported monomeric building block are connected by a peroxide (O22-) or a pair of fluoride (2 x F-) bridges. The nature of the bridge determines the distance between the metal ion dipoles leading to a dipolar coupling in the peroxido bridged compound of only ca. 70% of that in the bis-fluorido bridged dimer. The sign of the overall coupling between the metals is antiferromagnetic for the peroxido bridged compound and ferromagnetic for the bis-fluorido bridged complex. This in turn influences the magnetisation dynamics. We compare the relaxation characteristics of the dimers with those of the previously reported monomeric building block. The relaxation dynamics for the bis-fluorido system are very fast. On the other hand, comparing the properties of the monomer, the peroxido bridged sample and the corresponding Y-doped sample show that the relaxation properties via a Raman process have very similar parameters. We show that a second dysprosium is important for either tuning or detuning the Single Molecule Magnet (SMM) properties of a system.

Single-Molecule Investigation of Load-Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin.

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native ß-cardiac myosin (ßM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for ßM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins.

Simultaneous profiling of the RNA targets of two RNA-binding proteins using TRIBE-STAMP.

RNA-binding proteins (RBPs) are central players in RNA homeostasis and the control of gene expression. The identification of RBP targets, interactions, and the regulatory networks they control is crucial for understanding their cellular functions. Traditional methods for identifying RBP targets across the transcriptome have been insightful but are limited by their focus on a single RBP at a time and their general inability to identify individual RNA molecules that are bound by RBPs of interest. Recently, we overcame these limitations by developing TRIBE-STAMP, a method which enables concurrent identification of the RNA targets of two RBPs of interest with single-molecule resolution. TRIBE-STAMP works by tagging desired RBPs with either the ADAR or APOBEC1 RNA editing enzymes and expressing them in cells, followed by RNA-seq. Subsequent computational identification of A-to-I and C-to-U editing events enables the simultaneous identification of the ADAR- and APOBEC1-fused RBP target RNAs, respectively. Here, we present a detailed protocol for TRIBE-STAMP, including considerations for fusion protein expression in cells and step-by-step computational analysis of sequencing data. TRIBE-STAMP is a simple and highly versatile approach for single-molecule identification of the targets of RBPs which enables unprecedented insights into the biological interplay between RBP pairs in cells.

Simultaneous probing of transcription, G-quadruplex, and R-loop.

DNA and RNA can form various non-canonical secondary structures, including G-quadruplex (G4) and R-loops. These structures are considered transcriptional regulatory elements due to their enrichment at regulatory regions. During transcription, G-rich sequences in the non-template strand promote R-loop formation in the DNA template strand. These R-loops induce G4 structures in the non-template DNA strand, further stabilizing them. Additionally, the high rG: dC base-pairing within the R-loop contributes to the stability of DNA/RNA hybridization. Our previous study investigated the interplay between G4s and R-loops and its impact on transcription. We employed two techniques to demonstrate transcription-mediated G4 and R-loop formation. The single-molecule method allows us to detect intricate details of transcription initiation, elongation, and co-transcriptional R-loop and G4 formation. It provides a high-resolution view of the dynamic processes involved in transcriptional regulation. As an orthogonal approach, a gel-based assay enables the detection of the transcription-mediated R-loops and the RNA product. We can measure the progressive formation of R-loop and total RNA produced from transcription by analyzing gel electrophoresis patterns. In summary, these techniques provide valuable insights into the non-canonical nucleic acid structures and their impact on gene expression.

Utilizing nuclear extracts to characterize protein: DNA interactions at the single molecule level.

Single molecule experiments are invaluable approaches to analyze the dynamics of protein-protein and protein-DNA interactions in real time. SMADNE, single molecule analysis of DNA binding proteins from nuclear extracts, is a new method that allows analysis of a fluorescently tagged overexpressed protein of interest near its native environment while still retaining the advantages of single molecule approaches. Having all the endogenous proteins found in the nucleus provides more biologically relevant information due to their interactions with the protein of interest. Examples of such include the ability for posttranslational modifications to occur, intrinsic stabilization factors, and high labeling efficacy of the protein of interest. Furthermore, having the capabilities to incorporate different DNA substrates and protein variants can elucidate information of the system in a more detailed manner. Finally, orthogonal labeling strategies allows determination of the order of assembly and disassembly of several proteins at sites of damage. This chapter will describe the methodologies, benefits, and applications of SMADNE.

Measuring protein stoichiometry with single-molecule imaging in Xenopus egg extracts.

In human cells, DNA double-strand breaks are rapidly bound by the highly abundant non-homologous end joining (NHEJ) factor Ku70/Ku80 (Ku). Cellular imaging and structural data revealed a single Ku molecule is bound to a free DNA end and yet the mechanism regulating Ku remains unclear. Here, we describe how to utilize the cell-free Xenopus laevis egg extract system in conjunction with single-molecule microscopy to investigate regulation of Ku stoichiometry during non-homologous end joining. Egg extract is an excellent model system to study DNA repair as it contains the soluble proteome including core and accessory NHEJ factors, and efficiently repairs double-strand breaks in an NHEJ-dependent manner. To examine the Ku stoichiometry in the extract system, we developed a single-molecule photobleaching assay, which reports on the number of stable associated Ku molecules by monitoring the intensity of fluorescently labeled Ku molecules bound to double-stranded DNA over time. Photobleaching is distinguishable as step decreases in fluorescence intensity and the number of photobleaching events indicate fluorophore stoichiometry. In this paper we describe sample preparation, experimental methodology, and data analysis to discern Ku stoichiometry and the regulatory mechanism controlling its loading. These approaches can be readily adopted to determine stoichiometry of molecular factors within other macromolecular complexes.

Quantitative Advantages of Corrosion Sensing Using Fluorescence, Microscopy, and Single-Molecule Detection.

The corrosion of metals and alloys is a fundamental issue in modern society. Understanding the mechanisms that cause and prevent corrosion is integral to saving millions of dollars each year and to ensure the safe use of infrastructure subject to the hazardous degrading effects of corrosion. Despite this, corrosion detection techniques have lacked precise, quantitative information, with industries taking a top-down, macroscale approach to analyzing corrosion with tests that span months to years and yield qualitative information. Fluorescence, a well-established optical method, can fill the niche of early-stage, quantitative corrosion detection and can be employed for both bulk and localized testing over time. The latter, fluorescence microscopy, can be pushed to greater levels of detail with single-molecule microscopy, achieving nanometer spatial and subsecond temporal resolutions of corrosion that allow for the extraction of dynamic information and kinetics. This review will present how fluorescence microscopy can provide researchers with a molecular view into the chemical mechanisms of corrosion at interfaces and allow for faster, quantitative studies of how to detect and prevent corrosion.

Extreme Anomalous Conductance Enhancement in Neutral Diradical Acene-like Molecular Junctions.

We achieve, at room temperature, conductance enhancements over 2 orders of magnitude in single molecule circuits formed with polycyclic benzoquinoidal (BQn) diradicals upon increasing molecular length by ∼5 Å. We find that this extreme and atypical anti-ohmic conductance enhancement at longer molecular lengths is due to the diradical character of the molecules, which can be described as a topologically nontrivial electronic state, and results in constructive interference between the frontier molecular orbitals. The distinct feature of the compounds studied here as molecular wires is that they are characterized by moderate diradical character in the neutral state, allowing for robust and facile measurements of their transport properties. We adapt the 1D-SSH model, originally developed to examine electronic topological order in linear carbon chains, to the polycyclic systems studied here and find that it captures the anti-ohmic trends in this molecular series. Specifically, our model reveals that the mechanism of conductance enhancement with length in polycyclic systems is constructive quantum interference between the frontier orbitals with nontrivial topology, which is present in acene-like, but not in linear, molecular systems. Importantly, we use our model to predict and experimentally validate that anti-ohmic trends can be engineered through synthetic adjustments of the diradical character of the acene-like molecules. Overall, we achieve extreme anti-ohmic enhancement and mechanistic insight into electronic transport in a class of materials that we identify here as promising candidates for creating highly conductive and tunable nanoscale wires.

Reusable Microfluidic Chambers for Single-Molecule Microscopy.

Maintaining a consistent environment in single-molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in nonspecific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments, as biotin can be readily incorporated into these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times by using photocleavable biotin (PC-biotin) and UV-light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5 min UV exposure of a specific wavelength. Nonoptimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality as assessed by the nonspecific binding of fluorescently labeled DNA and protein samples and the recovery of the DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

Super-Resolution Surface-Enhanced Raman Scattering: Perspectives on the Past, Present, and Future.

Super-resolution surface-enhanced Raman scattering (SERS) allows researchers to overcome the resolution limit of far field optical microscopy and peer into electromagnetic hot spots with nanoscale resolution. By localizing the signal from single (or few) molecules on the surface of plasmonic nanoparticle aggregates, relationships between the spatial origin of the SERS signal, local electromagnetic field enhancements, and SERS intensity can be determined. This Perspective describes the successes and challenges of super-resolution SERS, from the earliest mapping of single-molecule SERS hot spots to the current state-of-the-art, while highlighting open questions and future opportunities to advance the field. Comparisons with fluorescence-based super-resolution imaging are discussed to help frame the unique challenges associated with performing SERS in the super-resolution regime.

Time Crystals from Single-Molecule Magnet Arrays.

Time crystals, a unique nonequilibrium quantum phenomenon with promising applications in current quantum technologies, mark a significant advance in quantum mechanics. Although traditionally studied in atom-cavity and optical lattice systems, pursuing alternative nanoscale platforms for time crystals is crucial. Here we theoretically predict discrete time crystals in a periodically driven molecular magnet array, modeled by a spin-S Heisenberg Hamiltonian with significant quadratic anisotropy, taken with realistic and experimentally relevant physical parameters. Surprisingly, we find that the time crystal response frequency correlates with the energy levels of the individual magnets and is essentially independent of the exchange coupling. The latter is unexpectedly manifested through a pulse-like oscillation in the magnetization envelope, signaling a many-body response. These results show that molecular magnets can be a rich platform for studying time-crystalline behavior and possibly other out-of-equilibrium quantum many-body dynamics.

Resolving Sulfation Posttranslational Modifications on a Peptide Hormone using Nanopores.

Peptide hormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on plant peptide hormones is, for example, essential for plant growth and development. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection. Nanopores can sensitively detect protein PTMs at the single-molecule level. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate the accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a powerful tool for identifying position-specific sulfation on peptide hormones and promising wider applications to identify protein PTMs.

From Molecular Electronics to Molecular Intelligence.

Molecular electronics is a field that explores the ultimate limits of electronic device dimensions by using individual molecules as operable electronic devices. Over the past five decades since the proposal of a molecular rectifier by Aviram and Ratner in 1974 ( Chem. Phys. Lett.1974,29, 277-283), researchers have developed various fabrication and characterization techniques to explore the electrical properties of molecules. With the push of electrical characterizations and data analysis methodologies, the reproducibility issues of the single-molecule conductance measurement have been chiefly resolved, and the origins of conductance variation among different devices have been investigated. Numerous prototypical molecular electronic devices with external physical and chemical stimuli have been demonstrated based on the advances of instrumental and methodological developments. These devices enable functions such as switching, logic computing, and synaptic-like computing. However, as the goal of molecular electronics, how can molecular-based intelligence be achieved through single-molecule electronic devices? At the fiftieth anniversary of molecular electronics, we try to answer this question by summarizing recent progress and providing an outlook on single-molecule electronics. First, we review the fabrication methodologies for molecular junctions, which provide the foundation of molecular electronics. Second, the preliminary efforts of molecular logic devices toward integration circuits are discussed for future potential intelligent applications. Third, some molecular devices with sensing applications through physical and chemical stimuli are introduced, demonstrating phenomena at a single-molecule scale beyond conventional macroscopic devices. From this perspective, we summarize the current challenges and outlook prospects by describing the concepts of "AI for single-molecule electronics" and "single-molecule electronics for AI".

Stabilization of the Compressed Planar Benzene Dianion in Inverse-Sandwich Rare Earth Metal Complexes.

For the first time, the capture of a planar antiaromatic benzene dianion in between two trivalent rare earth (RE) metal cations, each stabilized by two guanidinate ligands, is reported. The synthesized inverse-sandwich complexes [{(Me3Si)2NC(NiPr)2}2RE]2(µ-ƞ6:ƞ6-C6H6), (RE = Y (1), Dy (2), and Er (3)) feature a remarkably planar benzene dianion, previously not encountered for any metal ion prone to low or absent covalency. The -2 charge localization at the benzene ligand was deduced from the results obtained by single-crystal X-ray diffraction analyses, spectroscopy, magnetometry, and Density Functional Theory (DFT) calculations. In the 1H NMR spectrum of the diamagnetic Y complex 1, the equivalent proton resonance of the bridging benzene dianion ligand is drastically shifted to higher field in comparison to free benzene. This and the calculated highly positive Nucleus-Independent Chemical Shift (NICS) values are attributed to the antiaromatic character of the benzene dianion ligand.  The crucial role of the ancillary guanidinate ligand scaffold in stabilizing the planar benzene dianion conformation was also elucidated by DFT calculations. Remarkably, the planarity of the benzene dianion originates from the stabilization of the π-type orbitals of the d-manifold and compression through its strong electrostatic interaction with the two REIII sites.

Memory effects of transcription regulator-DNA interactions in bacteria.

Memory effect refers to the phenomenon where past events influence a system's current and future states or behaviors. In biology, memory effects often arise from intra- or intermolecular interactions, leading to temporally correlated behaviors. Single-molecule studies have shown that enzymes and DNA-binding proteins can exhibit time-correlated behaviors of their activity. While memory effects are well documented and studied in vitro, no such examples exist in cells to our knowledge. Combining single-molecule tracking (SMT) and single-cell protein quantitation, we find in living Escherichia coli cells distinct temporal correlations in the binding/unbinding events on DNA by MerR- and Fur-family metalloregulators, manifesting as memory effects with timescales of ~1 s. These memory effects persist irrespective of the type of the metalloregulators or their metallation states. Moreover, these temporal correlations of metalloregulator-DNA interactions are associated with spatial confinements of the metalloregulators near their DNA binding sites, suggesting microdomains of ~100 nm in size that possibly result from the spatial organizations of the bacterial chromosome without the involvement of membranes. These microdomains likely facilitate repeated binding events, enhancing regulator-DNA contact frequency and potentially gene regulation efficiency. These findings provide unique insights into the spatiotemporal dynamics of protein-DNA interactions in bacterial cells, introducing the concept of microdomains as a crucial player in memory effect-driven gene regulation.

This Microtubule Does Not Exist: Super-Resolution Microscopy Image Generation by a Diffusion Model.

Generative models, such as diffusion models, have made significant advancements in recent years, enabling the synthesis of high-quality realistic data across various domains. Here, the adaptation and training of a diffusion model on super-resolution microscopy images are explored. It is shown that the generated images resemble experimental images, and that the generation process does not exhibit a large degree of memorization from existing images in the training set. To demonstrate the usefulness of the generative model for data augmentation, the performance of a deep learning-based single-image super-resolution (SISR) method trained using generated high-resolution data is compared against training using experimental images alone, or images generated by mathematical modeling. Using a few experimental images, the reconstruction quality and the spatial resolution of the reconstructed images are improved, showcasing the potential of diffusion model image generation for overcoming the limitations accompanying the collection and annotation of microscopy images. Finally, the pipeline is made publicly available, runnable online, and user-friendly to enable researchers to generate their own synthetic microscopy data. This work demonstrates the potential contribution of generative diffusion models for microscopy tasks and paves the way for their future application in this field.