Single-molecule Level Research Articles

Single Molecule Kinetic Fingerprinting of Glycans on IgA1 Antibodies.

Immunoglobulin A (IgA) nephropathy is the most common form of primary glomerulonephritis and is triggered by damage to glomeruli from deposition of complexes formed between glycosylated IgA1 antibodies that are "galactose-deficient" and antibodies directed to these aberrant proteins. Currently, galactose deficiencies are detected with ensemble measurements, e.g., via mass spectrometry or liquid chromatography, which only measure average glycan-IgA1 ratios, but cannot resolve heterogeneity of O-glycosylation between different IgA1 populations. To resolve these differences at the single molecule level, we developed an assay to detect the glycosylation state of individual IgA1 using single molecule fluorescence microscopy. By using fluorescence resonance energy transfer (FRET), high concentrations of fluorescently labeled probes with low binding rates can be employed to observe the binding of protein probes to surface adhered target molecules and obtain their kinetic fingerprints. We measured the binding and unbinding rates of jacalin (a lectin binding to O-linked glycans) to individual IgA1 molecules on a glass surface. Adding galactose decreased binding, which demonstrated that the jacalin probe binds specifically to O-linked glycans on the hinge region of IgA1. This result is a first step toward using kinetic fingerprinting to sequence glycans on IgA1.

Multidimensional third-generation sequencing of modified DNA bases allows interrogation of complex biological systems

DNA exists biologically as a highly dynamic macromolecular complex subject to myriad chemical modifications that alter its physiological interpretation, yet most sequencing technologies only measure Watson-Crick base pairing interactions. Third-generation sequencing technologies can directly detect novel and modified bases, yet the difficulty and cost of training these techniques for each novel base has so far limited this potential. Here, we present a method based on barcoded split-pool synthesis to generate reference standard oligonucleotides allowing novel base sequencing. Using novel base detection, we perform multidimensional sequencing to retrieve information, both physiologically stored and experimentally encoded, from DNA, allowing us to characterize the preferential replication of deleterious mitochondrial genome mutations, the infection dynamics of a host-pathogen model, and the effect of chemotherapy on cancer cell DNA at the single molecule level. The low cost and experimental simplicity of this method make this approach widely accessible to the research community, enabling complex experimental interrogation across the biological sciences.

A high-throughput single-molecule platform to study DNA supercoiling effect on protein–DNA interactions

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA–protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced physiological levels of negative or positive supercoiling in these plasmids using gyrase or reverse gyrase, respectively. By comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR–Cas9 and the mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed that both negative and positive DNA supercoiling enhance the binding interaction between MutS and a mismatched base pair but do not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also open new avenues for exploring the intricate dynamics of protein–DNA interactions under the influence of supercoiling.

Circularly Polarized Luminescence and Photoinduced Spin Polarization in Helicene-Bis-TEMPO Diradicals.

Readily accessible high spin excited states are highly coveted due to their potential use in spintronic and quantum sensing applications. Adding a chiral twist to this problem potentially allows for control of the emergent spin polarization through the helicity of the system. Herein we report the realization of a novel, chiral, diradical system, H6-bis-TEMPO, consisting of two TEMPO radicals bridged by a chiral carbo[6]helicene, which after photoexcitation of the helicene core generates a weakly coupled three-spin system comprising the helicene triplet (S = 1) and the two TEMPO radicals (S = 1/2). Time-resolved absorption and theoretical calculations help explain the specific photophysics of this system, while time-resolved EPR shows that the TEMPO radicals become spin polarized once the photoexcited spin intermediate has decayed. Comparison with a nonradical bis-dibenzoate capped helicene H6-bis-BENZ experimentally validates the results. This work constitutes a first step toward the realization of molecular systems able to generate spin polarization through helical chirality at the single-molecule level. Moreover, the helically chiral TEMPO persistent radical showscircularly polarized luminescence.

Enzymatic Reactions Dictated by the 2D Membrane Environment.

The cell membrane is a critical component of cellular architecture, serving not only as a physical barrier enclosing the cytosol but also as a dynamic platform for various biochemical reactions. Due to the unique two-dimensional and fluidic environment of the membrane, reactions that occur on its surface are subject to specific physical constraints. While membrane-mediated reactions are known to play key roles in cellular regulation, their advantages and limitations remain inadequately explored. In this study, we reconstitute a classic proteolytic cleavage reaction at the membrane interface, designed for the real-time kinetic analysis down to the single-molecule level. By systematically altering the enzyme-membrane affinity, we examined enzyme-substrate interactions under various conditions. Our findings reveal that while the membrane environment significantly enhances enzymatic turnover rate, it also imposes diffusion limitations that immediately reduce this turnover rate over time. By adjusting the enzyme's membrane affinity to an intermediate level, we enable the enzyme to "hop" on the membrane surface, overcoming these diffusion constraints and sustaining high enzymatic turnover rate with faster kinetics. These results highlight the dual role of the membrane environment in regulating biochemical reactions, balancing enhanced reactivity with physical limitations. Moreover, the ability to dynamically tune membrane affinity to optimize reactions underscores the cell's capacity to regulate enzymatic processes efficiently. This study provides critical insights into the role of the cell membrane in biochemical reactions and offers a broadly applicable framework for understanding membrane-associated interactions in biological systems.

Photoemission spectroscopy of organic molecules using plane wave/pseudopotential density functional theory and machine learning: A comprehensive and predictive computational protocol for isolated molecules, molecular aggregates, and organic thin films.

Photoemission measurements in the gas phase at low pressure have enabled the exploration of the intricate relationship between electronic and structural properties at the single-molecule level. Experimental data collected from isolated molecules, free from interactions with other species, have provided an ideal testing ground for developing abinitio simulations capable of interpreting and predicting photoemission spectra. In particular, accurate computational methods for determining atom- and site-specific core ionization binding energies (BEs) facilitate experimental data interpretation, enabling the assignment of contributions from non-equivalent atoms of the same species, even when spectral features remain unresolved due to molecular structure. In this context, we have developed, extensively tested, and made widely available a computational protocol based on plane wave/pseudopotential density functional theory (PW-DFT) within a ΔSCF framework to predict x-ray photoemission spectra (XPS) of isolated molecules. Moreover, we have preliminarily tested and demonstrated the applicability of the same method to large molecular aggregates and thin molecular films deposited on inorganic substrates. The protocol has been assessed using a representative set of semilocal and hybrid density functionals with increasing fractions of Hartree-Fock exact exchange (EXX), including PBE, B3LYP (20% EXX), HSE (range-separated with 25% EXX at short range), and BH&HLYP (50% EXX). As a benchmark, we have also employed the equation-of-motion coupled-cluster method with single and double excitations. Our protocol has been validated across a diverse range of molecular classes-including aromatic, heteroaromatic, and aliphatic compounds; drugs; and biomolecules-demonstrating high accuracy and robustness, even when using semilocal DFT. In addition, valence photoemission measurements complement core photoemission by providing insights into delocalized and π-conjugated molecular orbitals. These measurements are particularly useful for studying chemical modifications in large molecules mediated by non-covalent interactions. Using the same set of density functionals, we have evaluated their capability to predict valence-shell ionization spectra, employing Kohn-Sham eigenvalues as estimators. Finally, our PW-DFT dataset of C1s, N1s, and O1s BEs has been used to train machine learning (ML) models for predicting XPS spectra of isolated organic molecules based on their structure. To ensure reproducibility and encourage the adoption of our protocol, we have made available a public repository containing pseudopotentials, input files for abinitio calculations, and datasets used for ML model training.

Accessing Single-Molecule Properties of Heptacene Using a Metal-Organic Framework.

Acenes, classic polycyclic aromatic hydrocarbons composed of linearly fused benzene rings, represent a model system for exploring the physical properties of 1D π-conjugated structures. Isolating individual molecules at the single-molecule level provides a means to investigate their intrinsic properties, which is typically done by dissolving in solutions. However, this approach becomes increasingly difficult to apply for higher acenes owing to their high insolubility and instability. Thus, the electronic structure of higher acenes has long been a subject of intense discussion. This paper introduces a method to encapsulate heptacene within a metal-organic framework (MOF) through in situ photochemical conversion of a precursor molecule. The transformation reaction of the precursor is significantly accelerated upon inclusion. This approach stabilizes otherwise unstable heptacene by suppressing undesirable side reactions through the spatial constraint. The bulk production of heptacene in an isolated state enables its exploration through various analytical techniques, providing insights into its single-molecule properties and leading to the first observation of its fluorescence. Moreover, experimental and theoretical studies reveal the electronic ground state of pristine heptacene.

Label-Free Single-Molecule Immunoassay.

Single-molecule immunoassay is a reliable technique for the detection and quantification of low-abundance blood biomarkers, which are essential for early disease diagnosis and biomedical research. However, current single-molecule methods predominantly rely on endpoint detection and necessitate signal amplification via labeling, which brings a variety of unwanted effects, like matrix effect and autofluorescence interference. This study introduces a real-time mass imaging-based label-free single-molecule immunoassay (LFSMiA). Featuring plasmonic scattering microscopy-based mass imaging, a 2-step sandwich assay format enables background reduction, minimization of matrix effect by dynamic tracking of single binding events, and fully leveraging real-time data for improved measurement precision through a Bayesian Gaussian process model, the LFSMiA enables ultra-sensitive and direct protein detection at the single-molecule level in neat blood sample matrices. LFSMiA measurement is demonstrated for interleukin-6 and prostate-specific antigen in buffer, undiluted serum, and whole blood with sub-femtomolar detection limits and eight logs of dynamic ranges. Moreover, comparable performance is achieved with an inexpensive miniaturized setup. To show its translational potential to clinical settings and point-of-care diagnostics, N-terminal pro-B-type natriuretic peptide is examined in patient whole blood samples using the LFSMiA and results in a strong linear correlation (r > 0.99) with standard clinical lab results.

Decoy DNA Protects Molecular Tension Probes from DNase Degradation.

DNA-based molecular probes are essential tools for visualizing and quantifying mechanotransduction at the single-molecule level. However, their application in live-cell environments is severely limited by DNase-mediated degradation, which shortens probe lifespan and introduces false-positive signals. Here, we present a decoy DNA strategy where an excess of unmodified double-stranded DNA competitively binds DNases, effectively preserving functional DNA probes. This approach extends probe stability from 1-2 h to beyond 24 h, substantially improving signal integrity in live-cell tension imaging. In contrast to structurally modified nucleic acids, decoy DNA can be readily applied to existing DNA probe systems, enabling seamless integration without the need for additional validation or calibration. This cost-effective and scalable strategy provides a generalizable framework for stabilizing DNA-based molecular tools in DNase-rich environments, enabling high-precision mechanobiology studies across diverse cell types and extended experiment durations.

Methoxy-Substituted Perylenediimide-Modified DNA for Enhanced Fluorescent Probes and Single-Nucleotide Discrimination.

Artificial DNA molecules functionalized with fluorescent dyes are useful for developing fluorescent chemosensors and molecular imaging tools. Fluorescent nucleic acids have been developed based on the excellent fluorescent properties of perylenediimide (PDI); however, the electron transfer quenching of PDI by purine bases limits the sequence design of fluorescent probes. In this study, to compensate for the disadvantages of PDI due to electron transfer quenching, we used PDI with a methoxy group introduced into the perylene ring (PO), synthesized PO-substituted nucleic acids, and investigated their fluorescence properties. The results showed that PO in the DNA exhibited longer-wavelength fluorescence than unsubstituted PDI and displayed strong fluorescence when surrounded by AT base pairs. We also found that PO exhibits a fluorescence on-off response to the hybridization reaction and selective quenching by guanine bases in the vicinity of PO, which could be applied to fluorescent biosensors for single nucleotide identification and nucleic acid detection. Because of the high photostability of PO, single-molecule measurements were feasible, allowing confirmation of PO's fluorescence switching behavior of PO in response to DNA conformational changes and the presence of guanine bases at the single-molecule level using fluorescence correlation spectroscopy (FCS).

Single-Molecule Imaging for Unraveling the Functional Diversity of 10-23 DNAzymes.

DNA-based enzymes, also known as DNAzymes, have opened new opportunities for signal generation and amplification in several fields including biosensing. However, biosensor performance can be hampered by heterogeneity in the catalytic activity of such DNAzymes, especially when relying on a limited number of molecules to generate signal. In this regard, single-molecule studies are essential to discern the behavior among such heterogeneous molecules otherwise masked by ensemble measurements. This work presents a novel methodology to study the 10-23 RNA-cleaving DNAzyme at the single-molecule level. By means of measuring the distance-sensitive efficiency of Förster Resonance Energy Transfer using alternating-laser excitation on a superresolution microscope, we determined the kinetics of individual DNAzymes in terms of substrate turnover, rates of different reaction steps, and changes in performance over time. Our results revealed that, despite high concentrations of the reaction cofactor (i.e., Mg2+), a maximum of only 70% of the DNAzymes are actively cleaving multiple substrate sequences; the DNAzyme molecules also showed a wide range of substrate turnover rates. Our findings shed new light on the functional diversity of DNAzymes and the importance of exploring sequence modifications to improve their catalytic performance. Ultimately, this work presents a technique to obtain time-dependent information, which could be easily implemented to study other types of enzymes or biomolecular interactions.

Studying the Effect of Receptors Clustering on Hyaluronic Acid Binding with CD44 and the Cell Entry of Hyaluronic Acid Targeting Nanodrugs at Single Molecule/Particle Level.

Receptor-ligand interactions on cell membrane play key roles in many physiological and pathological processes, especially in the nanodrug targeted delivery system. Hyaluronic acid (HA) is the main ligand for binding CD44 to modulate the targeted delivery of nanodrugs; however, the corresponding receptor clustering mechanism remains unclear. The differential effects of HA on CD44 clustering are closely associated with the number of disaccharide units. Herein, the binding dynamic parameters between CD44 and HA containing various disaccharide units were analyzed at the single-molecule level. Then, the clustering effect induced by HA containing various disaccharide units was evaluated and the optimal clustering effect was verified. Furthermore, the clustering effect on cell entry dynamic parameters of HA targeting nanodrugs was assessed based on different cell lines at the single particle level. These results demonstrate that the clustering effect will enhance the entry cell efficiency of HA targeting nanodrugs, and the effect is more obvious on the cell line with low expression level of CD44. This study offers a new way to evaluate the cell membrane receptor clustering and the corresponding effect on cellular uptake, which will provide potential strategy for designing appropriate targeting nanodrugs with high delivery efficiency tailored to different cancers.

Decoding stimulus-specific regulation of promoter activity of p53 target genes

The tumor suppressor p53 plays a crucial role in maintaining genome integrity in response to exogenous or endogenous stresses. The dynamics of p53 activation are stimulus- and cell type-dependent and regulate cell fate. Acting as a transcription factor, p53 induces the expression of target genes involved in apoptosis, cell cycle arrest and DNA repair. However, transcription is not a deterministic process, but rather occurs in bursts of activity and promoters switch stochastically between ON and OFF states, resulting in substantial cell-to-cell variability. Here, we characterized how stimulus-dependent p53 dynamics are converted into specific gene regulation patterns by inducing diverse forms of DNA damage ranging from ionizing and UV radiation to clinically relevant chemotherapeutics. We employed single molecule fluorescence in-situ hybridization (smFISH) to quantify the activity of target gene promoters at the single-cell and single-molecule level. To analyse this comprehensive data set, we developed a new framework for determining parameters of stochastic gene expression by Bayesian inference. Using this combined theoretical and experimental approach, we revealed that features of promoter activity are differentially regulated depending on the target gene and the nature and extent of the DNA damage induced. Indeed, stimulus-specific stochastic gene expression is predominantly regulated by promoter activation and deactivation rates. Interestingly, we found that in many situations, transcriptional activity was uncoupled from the total amount of p53 and the fraction bound to DNA, highlighting that transcriptional regulation by p53 is a multi-dimensional process. Taken together, our study provides insights into p53-mediated transcriptional regulation as an example of a dynamic transcription factor that shapes the cellular response to DNA damage.

Protein Sequencing with Single Amino Acid Resolution Discerns Peptides That Discriminate Tropomyosin Proteoforms.

Protein variants of the same gene─proteoforms─can have high molecular similarity yet exhibit different biological functions. Thus, the identification of unique peptides that unambiguously map to proteoforms can provide crucial biological insights. In humans, four human tropomyosin (TPM) genes produce similar proteoforms that can be challenging to distinguish with standard proteomics tools. For example, TPM1 and TPM2 share 85% sequence identity with amino acid substitutions that play unique roles in muscle contraction and myopathies. In this study, we evaluated the ability of the recently released Platinum single-molecule protein sequencer to detect proteoform-informative peptides. Platinum employs fluorophore-labeled recognizers that reversibly bind to cognate N-terminal amino acids (NAAs), enabling polypeptide sequencing within nanoscale apertures of a semiconductor chip that can accommodate single peptide molecules. As a proof of concept, we evaluated the ability of Platinum to distinguish three main types of proteoform variation: paralogue-level, transcript-level, and post-translational modification (PTM). We distinguished paralogous TPM1 and TPM2 peptides differing in a single isobaric residue (leucine/isoleucine). We also distinguished tissue-specific TPM2 spliceforms. Notably, we found that a phosphotyrosine-modified peptide displayed a reduced recognizer affinity for tyrosine, showing sensitivity to PTMs. This study paves the way for the targeted detection of proteoform biomarkers at the single molecule level.

Modulation of SARS-CoV-2 spike binding to ACE2 through conformational selection.

The first step of SARS-CoV-2 infection involves the interaction between the viral trimeric spike protein (S) and the host angiotensin-converting enzyme 2 (ACE2). The receptor-binding domain (RBD) of S adopts two conformations: open and closed, respectively accessible and inaccessible to ACE2. Although these changes surely affect ACE2 binding, a quantitative description of the underlying mechanisms has remained elusive. Here we visualize RBD opening and closing using high-speed atomic force microscopy, gaining access to the corresponding transition rates. We also probe the S/ACE2 interaction at the ensemble level with biolayer interferometry and at the single-molecule level with atomic force microscopy and magnetic tweezers, evidencing that RBD dynamics hinder ACE2 binding but have no effect on unbinding. The resulting modulation is quantitatively predicted by a conformational selection model in which each S protomer behaves independently. Our work thus reveals a molecular mechanism by which RBD accessibility and binding strength can be tuned separately, providing hints to better understand the joint evolution of immune evasion and infectivity.

Ionic Liquid Accelerates Electrochemically Driven Single-Molecule Oxidative Coupling.

Molecular adsorption covers a broad spectrum of chemical processes and device fabrications at solid/liquid interfaces. The unraveling of the underlying mechanisms relies on regulating molecular adsorption. However, achieving such regulation at the single-molecule level remains a challenge. Herein, we utilized a combined ionic liquid and scanning tunneling microscope break junction methods to tune single 4-(pyridin-4-yl)aniline molecules gradually from the flat configuration to the upright one and found that only the upright configuration can trigger the oxidative coupling. Experimental and theoretical findings demonstrated that the ionic liquid reduced the electron density of the Au electrode, giving rise to the evolution of the interaction between molecule and electrode from the Au-π dominated coupling to the Au-σ dominated one. This work will inspire the exploration of strategies that can control the molecular assembly, accelerate the chemical reaction, and promote the fabrication of organic devices.

DNA Hanger: Surface-Minimized Single-Molecule Immunoassay Platform.

A novel single-molecule immunoassay platform, termed DNA Hanger, is developed to address the limitations of conventional surface-based assays. By suspending biotinylated λ-phage DNA across microfabricated quartz barriers, this method enables high-specificity protein detection with minimal nonspecific binding. DNA Hanger significantly reduces background signals, achieving nonspecific binding rates as low as one protein per 236µm of DNA. Quantification of mNeonGreen-tagged human poly(A)-binding protein C1 (mNG-PABP) and single-molecule fluorescence-linked immunosorbent assay (FLISA) of human tumor necrosis factor α (TNF-α) demonstrates the assay's specificity and sensitivity at the single-molecule level, with a detection limit of 0.90 pM in buffer, 38-fold lower than that of conventional FLISA, and 20.6 pM in 70% fetal bovine serum, an 8-fold improvement. DNA Hanger also enables the detection and quantification of endogenous TNF-α in human serum, highlighting its clinical potential. The DNA Hanger assay eliminates the need for surface blocking and simplifies workflow, resulting in completing the immunoassay process within 1 hour. DNA Hanger offers broad applicability for biomolecular interaction studies and clinical diagnostics.

Spin Manipulation of Single Nitroxide Radical on Au(111) by Selective Coordination.

Organic radicals are promising candidates for constructing molecule-based magnetic materials. Despite the achievements of various radical ligand-containing complex materials, tuning the spin state of organic radicals at the single-molecule level on surfaces remains a challenge. In this study, the spin state of the DPBIN molecule, a derivative of 1,4-di(pyridine-3-yl)benzene bearing two imino-nitroxide radicals in para positions, is tuned by selective coordination with Au, Ni, and Fe centers on the Au(111) surface. The DPBIN molecules provide nitroxide oxygen atoms as ligand atoms in the Au and Fe coordination structures, where the spin of the DPBIN molecules is quenched. By contrast, the nitroxide oxygen atoms are not involved in coordination with Ni atoms; thus, the spin is preserved in this structure. Scanning tunneling microscopy (STM) and spectroscopy (STS) are employed to characterize the geometric structures and spin states of these coordination structures at the atomic level. Interestingly, this spin-manipulation method demonstrates a broader applicability to other metal coordination systems. This research deepens our understanding of the effect of selective coordination on radical spins.

Nanopore-Based Single-Molecule Logic Unit (sMOLU).

DNA computing has recently advanced from theoretical computations to a wide variety of applications. Single-molecule DNA computing combined with nanopore technology offers a unique approach to real-time molecular computation. A single-molecule logic unit (sMOLU) is developed that uses α-hemolysin nanopores to implement a DNA-based AND logic gate within lipid bilayers. This molecular system leverages a three-way DNA junction immobilized within a nanopore on a lipid membrane, enabling enzyme-specific cleavage and signal amplification in giant unilamellar vesicles (GUVs). Fluorescence and electrophysiological measurements confirm precise logic gate operations at the single-molecule level. The sMOLU platform demonstrates the potential for advancing nanoscale computational systems with precise molecular analysis capability by integrating DNA computing and nanopore technologies.

Single-molecule parallel analysis for rapid exploration of sequence space.

Single-molecule fluorescence techniques have been successfully applied to uncover the structure, dynamics and interactions of DNA, RNA and proteins at the molecular scale. While the structure and function of these biomolecules are imposed by their sequences, single-molecule studies have been limited to a small number of sequences due to constraints in time and cost. To gain a comprehensive understanding on how sequence influences these essential biomolecules and the processes in which they act, a vast number of sequences have to be probed, requiring a high-throughput parallel approach. To address this need, we developed SPARXS: single-molecule parallel analysis for rapid exploration of sequence space. This platform enables simultaneous profiling of millions of molecules, covering thousands of distinct sequences, at the single-molecule level by coupling single-molecule fluorescence microscopy with next-generation high-throughput sequencing. Here we describe how to implement SPARXS and give examples from our study into the effect of sequence on Holliday junction kinetics. We provide a detailed description of sample and library design, single-molecule measurement, sequencing, coupling of sequencing and single-molecule fluorescence data, and data analysis. The protocol requires experience with single-molecule fluorescence microscopy and a basic command of Python to use our Papylio package for SPARXS data analysis. Familiarity with the underlying principles of Illumina sequencing is also beneficial. The entire process takes ~1-2 weeks and provides a detailed quantitative picture of the effect of sequence on the studied process.