Single-molecule Level Research Articles

Review of: "Plasmonic-based nanosensors have received considerable attention due to their extreme sensitivity even at the single molecule level"

Review of: "Plasmonic-based nanosensors have received considerable attention due to their extreme sensitivity even at the single molecule level"

Profiling the epigenome using long-read sequencing.

The advent of single-molecule, long-read sequencing (LRS) technologies by Oxford Nanopore Technologies and Pacific Biosciences has revolutionized genomics, transcriptomics and, more recently, epigenomics research. These technologies offer distinct advantages, including the direct detection of methylated DNA and simultaneous assessment of DNA sequences spanning multiple kilobases along with their modifications at the single-molecule level. This has enabled the development of new assays for analyzing chromatin states and made it possible to integrate data for DNA methylation, chromatin accessibility, transcription factor binding and histone modifications, thereby facilitating comprehensive epigenomic profiling. Owing to recent advancements, alternative, nascent and translating transcripts can be detected using LRS approaches. This Review discusses LRS-based experimental and computational strategies for characterizing chromatin states and highlights their advantages over short-read sequencing methods. Furthermore, we demonstrate how various long-read methods can be integrated to design multi-omics studies to investigate the relationship between chromatin states and transcriptional dynamics.

Single-Molecule Tracking and Super-Resolution Microscopy Unveil Actin-Driven Membrane Nanotopography Shaping Stable Integrin Adhesions in Developing Tissue.

Single molecule tracking and super-resolution microscopy of integrin adhesion proteins and actin in developing Drosophila muscle attachment sites reveals that nanotopography triggered by Arp2/3-dependent actin protrusions promotes stable adhesionformation. The nanodomains formed during this process confine the diffusion of integrins and promote their immobilization. Spatial confinement is also applied to the motion of actin filaments, resulting in enhanced mechanical connection with the integrin adhesion complex. Fabricated nano-structured surfaces mimicking the nanotopography observed in livingtissue are able to recapitulate the formation of these adhesions in isolated muscle cells and the confinement of integrin diffusion. These results emphasize the importance of geometrical regulation of tissue morphogenesis at a single molecule level.

Impact of Structural Subtleties on Chirality Induction and Amplification in Trizinc(II)Porphyrin Trimers.

Herein, we report the precise control of molecular to supramolecular chirality induction at the single-molecule level just upon subtle modification in an achiral 'nano-size' trizinc(II) porphyrin trimer. A slight variation in the projection of the substituent at the periphery of the central porphyrin unit in a porphyrin trimer (host) resulted in pronounced changes in the interchromophoric arrangement, leading to distinct 'open' and 'closed' conformations. While 'open' form generates 'monomeric' complex with low CD amplitude, 'closed' form produces exclusive 'polymer' with large, amplified CD signal with opposite sign due to stronger intermolecular excitonic coupling. Also, the sign of the CD couplets is just opposite between R and S substrates for both polymer and monomer which suggest that the chirality is predominantly governed by the stereogenic projection of the chiral center. X-ray structures of the host and polymer have been reported here. Crystallographic characterization offers a detailed perspective on the structural and geometrical transformations in the polymer, enabling a systematic understanding of its optical properties. DFT and TD-DFT calculations strongly corroborate with the experimental findings.

Preparation of a Substrate for Studying Nucleosomes on Single Molecules of DNA.

DNA tightropes are a powerful single-molecule microscopy platform for the study of protein interactions with DNA on a single-molecule level. DNA molecules are suspended between two beads, granting full 3D access for a protein to bind and its interactions to be studied. Furthermore, tightropes can be custom-designed, permitting the study of protein interactions that are sequence- or substrate-dependent such as base mismatches or lesions. In this chapter, we describe the methodology required to generate novel nucleosome tightropes from plasmid to flow chamber. We hope this technique will enable researchers to study DNA-binding protein interactions with chromatinized DNA shedding more light on the intricate processes happening within the context ofchromatin.

Isolation and Analysis of Rare Enzymatic Events with Multiplex Flow Magnetic Tweezers.

Our understanding of biomolecular dynamics has been revolutionized with the advent of techniques that enable the manipulation of forces and torques at the single-molecule level. However, the characterization of rare intermediates has proven challenging due to limited throughput. In this chapter, we present a method that dramatically enhances the throughput of force spectroscopy measurements with topological control. The method allows for routine imaging of tens of thousands of individual molecules undergoing millions of reaction cycles in parallel. The improvement in throughput enables the discovery of rare enzymatic events. Here, we describe the experimental procedures for the observation and analysis of supercoiling dynamics by DNA gyrase. To efficiently quantify diverse dynamic behaviors and rare events, we introduce a software platform with an incorporated automated feature classification pipeline. This method and accompanying software can be freely adapted for investigations into a wide array of complex, multistep enzymatic pathways where the characterization of rare intermediates has been hindered by limited throughput.

Toward nanofabrication of SERS substrates with two-photon polymerization.

Surface-enhanced Raman spectroscopy (SERS) has shown its ability to characterize biological substances down to a single-molecule level without a specific biorecognition mechanism. Various nanofabrication technologies enable SERS substrate prototyping and mass manufacturing. This study reports a complete cycle of design, fabrication, prototyping, and metrology of SERS substrates based on two-photon polymerization (2PP). Highly controllable direct laser writing allows the fabrication of individual nanopillars with up to an aspect ratio of 4. The developed SERS substrates show up to 106 Raman signal enhancement, comparable to commercial substrates. Moreover, the rapid prototyping of the 2PP-printed SERS substrates takes from a minute to less than 2 hours, depending upon the nano-printing approach and aspect ratio requirements. The process is well-controlled and reproducible for achieving a uniform distribution of nanostructure arrays, allowing the SERS substrates to be used for a broad range of applications and the characterization of different molecules.

Unravelling molecular mechanobiology using DNA-based fluorogenic tension sensors.

Investigations of the biological system have revealed many principles that govern regular life processes. Recently, the analysis of tiny mechanical forces associated with many biological processes revealed their significance in understanding biological functions. Consequently, this piqued the interest of researchers, and a series of technologies have been developed to understand biomechanical cues at the molecular level. Notable techniques include single-molecule force spectroscopy, traction force microscopy, and molecular tension sensors. Well-defined double-stranded DNA structures could possess programmable mechanical characteristics, and hence, they have become one of the central molecules in molecular tension sensor technology. With the advancement of DNA technology, DNA or nucleic acid-based robust tension sensors offer the possibility of understanding mechanobiology in the bulk to single-molecule level range with desired spatiotemporal resolution. This review presents a comprehensive account of molecular tension sensors with a special emphasis on DNA-based fluorogenic tension sensors. Along with a detailed discussion on irreversible and reversible DNA-based tension sensors and their application in super-resolution microscopy, a discussion on biomolecules associated with cellular mechanotransduction and key findings in the field are included. This review ends with an elaborate discussion on the current challenges and future prospects of molecular tension sensors.

Non-sticky SiNx nanonets for single protein denaturation analysis.

Proteins play crucial roles in nearly all biological activities, with their functional structures deriving from stable folded conformations. Protein denaturation, induced by chemical and physical agents, is a complex process where proteins lose their stable structures, thereby impairing their biological functions. Characterizing protein denaturation at the single-molecule level remains a significant challenge. In this study, we developed non-adhesive silicon nitride nanonets coated with polyethylene glycol to capture individual proteins. We utilized these nanonets to investigate the denaturation of ovalbumin induced by guanidine hydrochloride (Gdn-HCl) and lead chloride. The entire denaturation and renaturation processes of a single ovalbumin molecule were monitored via ionic current measurements through the nanonets. These non-sticky nanonets offer a versatile tool for real-time studies of structural changes during protein denaturation.

Study on the SERS Effect and Mechanism of Vertically Oriented MoS<sub>2</sub> Nanosheet Composite with Ag Substrate

Surface enhanced Raman spectroscopy (SERS) can provide rich molecular structure information with ultra-sensitive, non-destructive, and rapid detection down to the single-molecule level. It has been widely applied in physics, chemistry, biomedicine, environmental science, material science and other fields. Combining the advantages of metals and 2D nanomaterials, various 2D/metal composite structures have been proposed for SERS. However, the contribution of 2D nanomaterials in Raman enhancement is often limited. In this work, vertically aligned MoS<sub>2</sub> nanosheet composite with silver nanoparticles (Ag NPs) was proposed for SERS detection. Large-area vertically aligned MoS<sub>2</sub> nanosheets, which were grown directly on molybdenum (Mo) foil using hydrothermal method, can effectively enhance molecular adsorption, light absorption, and provide dual electromagnetic and chemical enhancement. Furthermore, annealing treatment of the MoS<sub>2</sub> nanosheets significantly improves the efficiency of charge transfer between Ag NPs and MoS<sub>2</sub>, thereby increasing the chemical contribution to SERS. The results demonstrate that the annealed MoS<sub>2</sub>/Ag substrate exhibits outstanding SERS performance, with a detection limit for R6G molecules as low as 10<sup>-12</sup> M, which is four orders of magnitude lower than that of the unannealed substrate. The enhancement factor (EF) is calculated to be approximately 1.08×10<sup>9</sup>, approaching the sensitivity required for single-molecule detection. Additionally, the substrate performs high signal reproducibility at low concentrations, enabling ultra-sensitive detection of pesticide residues in aquatic products.

Self-Assembly of Human Fibrinogen into Microclot-Mimicking Antifibrinolytic Amyloid Fibrinogen Particles.

Recent clinical studies have highlighted the presence of microclots in the form of amyloid fibrinogen particles (AFPs) in plasma samples from Long COVID patients. However, the clinical significance of these abnormal, nonfibrillar self-assembly aggregates of human fibrinogen remains debated due to the limited understanding of their structural and biological characteristics. In this study, we present a method for generating mimetic microclots in vitro. Using this approach, the self-assembly process, structural organization of AFPs, and their interactions with human plasma components were elucidated. The amyloid transition of fibrinogen occurs under acidic conditions within a pH range of 2.3-3.2. Well-dispersed amyloid oligomers of fibrinogen, ranging in size from 1 to 5 μm, can be prepared at pH 2.8 after 1 h of incubation. We tracked the dynamic self-assembly process at the single-molecule level using high-speed atomic force microscopy (HS-AFM). The arrangement of amyloid oligomers manifests as well-ordered, stacked nanodomains with striped patterns, growing perpendicular to the primary axis of the fibrinogen monomer. Upon transfer to physiological solution conditions or human plasma, these amyloid oligomers further aggregate into nonfibrillar structures at the micrometer scale, resembling the microclots observed in the bloodstream of Long COVID patients. Notably, these AFPs exhibit characteristics consistent with microclots, including positive staining in thioflavin T (ThT) assays and resistance to fibrinolysis. Proteomic analysis suggests that AFPs interact with various components of human plasma and have an enhanced binding affinity with complement C3 compared to native fibrinogen. This study enables the in vitro preparation of mimetic microclots exhibiting amyloid features. It is anticipated to facilitate further researches on the mechanisms, detection, and treatment of diseases associated with fibrinogen amyloidogenesis.

Application and Development of Biomimetic Solid-State Nanopore in Biosensing Technique

Biomimetic solid-state nanopore is a nano-level technology, which can be effectively used for many detection work, including DNA sequencing. The design of biomimetic solid-state nanopore is inspired by biological ion channels. Biomimetic solid-state nanopore displays many advantages. It can increase the processing characteristics while it can also ensure the performance of similar biological ion channels. It also exhibits controllable surface chemical properties, making biomimetic solid nanopores have more application space. When compared with traditional biosensors, biomimetic solid nanopores have many advantages such as low price, high sensitivity and high specificity. At the same time, biomimetic solid-state nanopores have full help for medicine, analytical chemistry, materials science, single molecule biophysics, single molecule in vitro diagnostics, and genetics. Therefore, in this work, the working mechanisms of several biosensors are introduced. In addition, the applications of nanopores in biosensing are outlined. Besides, the circuit design schemes for improving the efficiency of nanopore has been discussed. In conclusion, the help of nanopores at the single-molecule level is undoubtedly huge, and it could help mankind to understand the nanoscale world.

End-to-end simulation of nanopore sequencing signals with feed-forward transformers.

Nanopore sequencing represents a significant advancement in genomics, enabling direct long-read DNA sequencing at the single-molecule level. Accurate simulation of nanopore sequencing signals from nucleotide sequences is crucial for method development and for complementing experimental data. Most existing approaches rely on predefined statistical models, which may not adequately capture the properties of experimental signal data. Furthermore, these simulators were developed for earlier versions of nanopore chemistry, which limits their applicability and adaptability to the latest flow cell data. To enhance the quality of artificial signals, we introduce seq2squiggle, a novel transformer-based, non-autoregressive model designed to generate nanopore sequencing signals from nucleotide sequences. Unlike existing simulators that rely on static k-mer models, our approach learns sequential contextual information from segmented signal data. We benchmark seq2squiggle against state-of-the-art simulators on real experimental R9.4.1 and R10.4.1 data, evaluating signal similarity, basecalling accuracy, and variant detection rates. Seq2squiggle consistently outperforms existing tools across multiple datasets, demonstrating superior similarity to real data and offering a robust solution for simulating nanopore sequencing signals with the latest flow cell generation. seq2squiggle is freely available on GitHub at: github.com/ZKI-PH-ImageAnalysis/seq2squiggle. Supplementary data are available at Bioinformatics online.

Millisecond Label-Free Single Peptide Detection and Identification Using Nanoscale Electrochromatography and Resistive Pulse Sensing.

We are developing a unique protein identification method that consists of generating peptides proteolytically from a single protein molecule (i.e., peptide fingerprints) with peptide detection and identification carried out using nanoscale electrochromatography and label-free resistive pulse sensing (RPS). As a step in realizing this technology, we report herein the nanoscale electrochromatography of model peptides using thermoplastic columns with surfaces engineered to identify peptides via their molecularly dependent mobility (i.e., time-of-flight, ToF). ToFs were elucidated using a dual in-plane nanopore sensor, which consisted of two in-plane nanopores placed on either end of the nanoelectrochromatography column. The surface of the nanocolumn, which consisted of poly(methyl methacrylate) (PMMA), was activated with an O2 plasma, creating surface carboxylic acid groups (-COOH) inducing a surface charge on the column wall as well as affecting its hydrophilicity. To understand scaling effects, we carried out microchip and nanochannel electrochromatography of the peptides labeled with an ATTO 532 reporter to allow for single-molecule tracking. Our results indicated that the apparent mobilities of the model peptides did not allow for their separation in a microchannel, but when performed in a nanocolumn, clear differences in their apparent mobilities could be observed especially when operated at high electric field strengths. We next performed label-free detection of peptides using the dual in-plane nanopore sensor with the two pores separated by a 5 μm (length) column with a 50 nm width and depth. When a single peptide molecule passed through an in-plane nanopore, the sensor read a pair of resistive pulses with a time difference equivalent to ToF. We identified the peptides by evaluating their ToF, normalized RPS current transient amplitude (ΔI/I0), and RPS peak dwell time (td). We could identify the model peptides with nearly 100% classification accuracy at the single-molecule level using machine learning with a single molecule measurement requiring <10 ms.

Single-Molecule Electrical Conductance in Z-form DNA:RNA.

Nucleic acids have emerged as new materials with promising applications in nanotechnology, molecular electronics, and biosensing, but their electronic properties, especially at the single-molecule level, are largely underexplored. The Z-form is an exotic left-handed helical oligonucleotide conformation that may be involved in critical biological processes such as the regulation of gene expression and epigenetic processes. In this work, the electrical conductance of individual Guanine Cytosine (GC)-rich DNA:RNA molecules is measured in physiological buffer and 2,2,2-Trifluoroethanol (TFE) solvent, corresponding to the natural (right-handed helix) A-form typical in DNA:RNA hybrids and the (left-handed) Z-form conformations, respectively. Single-molecule conductance measurements are performed using the Scanning Tunneling Microscopy (STM)-assisted break-junction method in the so-called "blinking" approach, recording the spontaneous formation of single-biomolecule junctions and performing statistical analysis of the signals. Circular Dichroism (CD) experiments and ab initio calculations are also done to rationalize the measured molecular conductivity with a simple structural and electronic model. These results show that the electrical conductivity of the Z-form is one order of magnitude lower than that of the more compact A-form. The longer molecular length and higher energy for the Highest Occupied Molecular Orbital (HOMO) of the Z-form account for the differences in single-molecule conductance observed experimentally.

Correlated Molecular Motion during Release of Residual Stress in Polymer Glassy Films.

Correlated molecular motion during the process of residual stress release in polymer glassy films is studied at the single-molecule level. Using poly(n-butyl methacrylate) (PnBMA) and poly(vinyl acetate) (PVAc) as the model polymers, thin films fabricated by spin-casting without thermal annealing were chosen as samples for investigation. Single-molecule fluorescence defocused microscopy was used to track the rotational motion of the fluorescent probes doped inside the polymer films. Under the activation effect of residual stress at experimental temperatures, the rotational motions of individual probes are discovered to be correlated a few degrees below the glass transition temperature (Tg), by analyzing the cross-correlation function of the rotational trajectories of different probes. Detailed investigations into the dependence on residual stress strength, intermolecular distance, probe-polymer interaction, and molecular orientation have been conducted. The results have revealed that the physical mechanism of the motion correlation is the randomization process from the state with preferred molecular orientation and presumably the polymer chain stretching.

Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.

The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.

Single-Molecule Multivalent Interactions Revealed by Plasmon-Enhanced Fluorescence.

Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling, which is also a current concept in drug design. Here, we use the high signals from plasmon-enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real time. We study mono-, bi-, and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency and introduce a step-binding model for binding kinetics relevant for structured macromolecules by including an experimentally extractable binding restriction term ω to quantify the effects from conformation, steric effects, and rigidity. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where the overall binding strength decreased with spacer length. For trivalent systems, increasing spacer length additionally activated binding in the trivalent state, giving insight into the design of multivalent drug or targeting moieties. By systematically changing the receptor density, we explored the binding super selectivity of the multivalent HJ at the single-molecule level. We find a polynomial behavior of the trivalent binding strength that clearly shows receptor-density-dependent selective binding. Interestingly, we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal on the position of the dye to observe binding dynamics during single multivalent binding events.

Mapping the Extended Ground State Reactivity Landscape of a Photoswitchable Molecule at a Single Molecular Level.

Photoswitchable molecules with structural flexibility can exhibit a complex ground state potential energy landscape due to the accessibility of multiple metastable states at merely low energy barriers. However, conventional bulk analytical techniques are limited in their ability to probe these metastable ground states and their relative energies. This is partially due to the difficulty of inducing changes in small molecules in their ground state, as they do not respond to external stimuli, such as mechanical force, unless they are incorporated into larger polymer networks. This hinders the understanding of ground-state reactivity and the associated dynamics. In this study, we leverage the "perturb-probe" capability of the single molecular break junction technique to explore the ground state 6π electrocyclization of a dithienylethene (DTE) derivative, a process traditionally achieved through electro- or photochromism. Our findings reveal that this reaction can also be triggered by mechanical force and an oriented electric field at the single-molecule level via ground state dynamics. We demonstrated that external perturbations could control the ground state reaction dynamics and steer the reaction trajectories away from constraints imposed by typical excited state dynamics. This strategy will thus offer access to a whole new dimension of single molecular electromechanical conversions and extend our knowledge of the ground state potential energy surface available to molecules under external force fields.

XYLAN O-ACETYLTRANSFERASE 6 promotes xylan synthesis by forming a complex with IRX10 and governs wall formation in rice.

Xylan, a pivotal polymer with diversified structures, is indispensable for cell wall integrity and contributes to plant growth and biomass recalcitrance. Xylan is synthesized by multi-enzyme complexes named xylan synthase complexes (XSCs). However, the biochemical mechanism of XSCs and the functions of core components within XSC remain unclear. Here, we report that rice (Oryza sativa) XYLAN O-ACETYLTRANSFERASE 6 (XOAT6) and the xylan synthase IRREGULAR XYLEM10 (IRX10) represent core components of the XSC, acting together to biosynthesize acetyl-xylans. Co-fractionation mass spectrometry and protein-protein interaction analyses revealed that IRX10 and XOAT6 physically interact within XSC, corroborated by similar xylan defects in xoat6 and irx10 mutants. Biochemical assays showed that XOAT6 is an O-acetyltransferase of the xylan backbone and facilitates chain polymerization catalyzed by IRX10. Fluorescence correlation spectroscopy further visualized the xylooligomer polymerization process at a single-molecule level. Solid-state NMR analysis, electron microscopy observations, and nanoindentation examinations identified the altered xylan conformation, disorganized cellulosic structure, and increased wall rigidity and cellulose accessibility in the mutants, leading to brittleness and improved saccharification efficiency. Our findings provide insights into the assembly of XSCs and xylan biosynthesis and offer a framework for tailoring xylans to improve crop traits and biomass.