Single-molecule Measurements Research Articles

Acceleration of DNA Replication of Klenow Fragment by Small Resisting ForceSupported by the National Natural Science Foundation of China (Grant Nos. 11674381, 21991133, 11774407, 11874415, 11874414, and 31770812), the Key Research Program on Frontier Science (Grant No. QYZDB-SSWSLH045), the National Key Research and Development Program of China (Grant No. 2016YFA0301500), the CAS Strategic Priority Research Program (Grant No. XDB37010100), and

DNA polymerases are an essential class of enzymes or molecular motors that catalyze processive DNA syntheses during DNA replications. A critical issue for DNA polymerases is their molecular mechanism of processive DNA replication. We have proposed a model for chemomechanical coupling of DNA polymerases before, based on which the predicted results have been provided about the dependence of DNA replication velocity upon the external force on Klenow fragment of DNA polymerase I. Here, we performed single molecule measurements of the replication velocity of Klenow fragment under the external force by using magnetic tweezers. The single molecule data verified quantitatively the previous theoretical predictions, which is critical to the chemomechanical coupling mechanism of DNA polymerases. A prominent characteristic for the Klenow fragment is that the replication velocity is independent of the assisting force whereas the velocity increases largely with the increase of the resisting force, attains the maximum velocity at about 3.8 pN and then decreases with the further increase of the resisting force.

MRI-derived g-ratio and lesion severity in newly diagnosed multiple sclerosis.

Myelin loss is associated with axonal damage in established multiple sclerosis. This relationship is challenging to study in vivo in early disease. Here, we ask whether myelin loss is associated with axonal damage at diagnosis by combining non-invasive neuroimaging and blood biomarkers. We performed quantitative microstructural MRI and single-molecule ELISA plasma neurofilament measurement in 73 patients with newly diagnosed, immunotherapy naïve relapsing–remitting multiple sclerosis. Myelin integrity was evaluated using aggregate g-ratios, derived from magnetization transfer saturation and neurite orientation dispersion and density imaging diffusion data. We found significantly higher g-ratios within cerebral white matter lesions (suggesting myelin loss) compared with normal-appearing white matter (0.61 versus 0.57, difference 0.036, 95% CI: 0.029–0.043, P < 0.001). Lesion volume (Spearman’s rho rs= 0.38, P < 0.001) and g-ratio (rs= 0.24, P < 0.05) correlated independently with plasma neurofilament. In patients with substantial lesion load (n = 38), those with higher g-ratio (defined as greater than median) were more likely to have abnormally elevated plasma neurofilament than those with normal g-ratio (defined as less than median) [11/23 (48%) versus 2/15 (13%), P < 0.05]. These data suggest that, even at multiple sclerosis diagnosis, reduced myelin integrity is associated with axonal damage. MRI-derived g-ratio may provide useful additional information regarding lesion severity and help to identify individuals with a high degree of axonal damage at disease onset.

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor.

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.

Single-Molecule Measurements Spatially Probe States Involved in Electron Transfer from CdSe/CdS Core/Shell Nanorods

Semiconductor nanorods with charge-accepting molecules adsorbed on their surfaces serve as model systems for solar energy conversion. An electron photoexcited from the valence band of the nanorod to a high-energy state in the conduction band will relax and transfer to a state in the molecule, producing a long-lived charge-separated state that facilitates charge extraction and thereby enables photochemical reactions. Characterizing the dynamics of the charge-separation process and the electronic states involved is essential for a microscopic understanding of photocatalysis involving these materials, but this information is obscured in ensemble measurements due to the random placement of molecules on the nanorod surfaces. Here, we show that measurements on individual CdSe/CdS core/shell nanorods functionalized by single methyl viologen molecules provide information about the distribution of electron-transfer rates from confined states in the nanorods to states in the molecules. By comparing this transfer-rate distribution to the predictions of a tight-binding model, we find that charge transfer most likely involves hot electrons in an excited conduction-band state, rather than electrons that have fully thermalized to the conduction-band edge. The ability to extract hot electrons from semiconductor nanocrystals may help enable energy-efficient photocatalysis, and the single-particle charge-transfer method may serve as a widely applicable tool to probe the spatial distribution of electronic states in nanocrystals.

Single-Molecule Methods for Investigating the Double-Stranded DNA Bendability.

The various DNA-protein interactions associated with the expression of genetic information involve double-stranded DNA (dsDNA) bending. Due to the importance of the formation of the dsDNA bending structure, dsDNA bending properties have long been investigated in the biophysics field. Conventionally, DNA bendability is characterized by innate averaging data from bulk experiments. The advent of single-molecule methods, such as atomic force microscopy, optical and magnetic tweezers, tethered particle motion, and single-molecule fluorescence resonance energy transfer measurement, has provided valuable tools to investigate not only the static structures but also the dynamic properties of bent dsDNA. Here, we reviewed the single-molecule methods that have been used for investigating dsDNA bendability and new findings related to dsDNA bending. Single-molecule approaches are promising tools for revealing the unknown properties of dsDNA related to its bending, particularly in cells.

Excited-State Symmetry Breaking in a Multiple Multipolar Chromophore Probed by Single-Molecule Fluorescence Imaging and Spectroscopy.

Excited-state symmetry breaking (ESB) has attracted much attention because it is often observed in symmetric multipolar chromophores designed as two-photon absorption/emission materials. Herein, we report an ensemble and single-molecule fluorescence imaging and spectroscopy investigation of ESB in hexakis[4-(p-dioctylaminostyryl)phenylethynyl]benzene(DB6), a two-photon absorber possessing a C6-symmetric π-D6 structure (π = hexaethynylbenzene, D = (p-dioctylaminostyryl)phenyl group) consisting of three equivalent D-π-D moieties. Ensemble and single-molecule measurements and theoretical calculations revealed that DB6 undergoes a photoabsorption process with two orthogonal transition dipole moments, whereas it fluoresces with a single transition dipole moment after one- or two-step ESB upon photoexcitation, depending on the environmental polarity. In nonpolar solvents and polymer films, one of the three D-π-D sites becomes planar, and the excited state is localized on this moiety: a [Dδ+-πδ--Dδ+]* quadrupolar state is formed. In polar solvents, the symmetry is further broken within the planarized D-π-D moiety, and the excited state is localized on one of the two D-π sites; i.e., a D-[πδ--Dδ+]* dipolar state is generated. Hence, DB6 can behave like a multichromophore with multiple emission sites in the molecule, which was demonstrated by stepwise photobleaching under photon antibunching conditions.

Single-molecule measurements reveal that PARP1 condenses DNA by loop stabilization.

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear enzyme that plays important roles in DNA repair, chromatin organization and transcription regulation. Although binding and activation of PARP1 by DNA damage sites has been extensively studied, little is known about how PARP1 binds to long stretches of undamaged DNA and how it could shape chromatin architecture. Here, using single-molecule techniques, we show that PARP1 binds and condenses undamaged, kilobase-length DNA subject to sub-piconewton mechanical forces. Stepwise decondensation at high force and DNA braiding experiments show that the condensation activity is due to the stabilization of DNA loops by PARP1. PARP inhibitors do not affect the level of condensation of undamaged DNA but act to block condensation reversal for damaged DNA in the presence of NAD+ Our findings suggest a mechanism for PARP1 in the organization of chromatin structure.

Direct imaging of single-molecule electrochemical reactions in solution.

Chemical reactions tend to be conceptualized in terms of individual molecules transforming into products, but are usually observed in experiments that probe the average behaviour of the ensemble. Single-molecule methods move beyond ensemble averages and reveal the statistical distribution of reaction positions, pathways and dynamics1-3. This has been shown with optical traps and scanning probe microscopy manipulating and observing individual reactions at defined locations with high spatial resolution4,5, and with modern optical methods using ultrasensitive photodetectors3,6,7 that enable high-throughput single-molecule measurements. However, effective probing of single-molecule solution chemistry remains challenging. Here we demonstrate optical imaging of single-molecule electrochemical reactions7 in aqueous solution and its use for super-resolution microscopy. The method utilizes a chemiluminescent reaction involving a ruthenium complex electrochemically generated at an electrode8, which ensures minimal background signal. This allows us to directly capture single photons of the electrochemiluminescence of individual reactions, and to develop super-resolved electrochemiluminescence microscopy for imaging the adhesion dynamics of live cells with high spatiotemporal resolution. We anticipate that our method will advance the fundamental understanding of electrochemical reactions and prove useful for bioassays and cell-imaging applications.

Using Weakly Supervised Deep Learning to Classify and Segment Single-Molecule Break-Junction Conductance Traces.

In order to design molecular electronic devices with high performance and stability, it is crucial to understand their structure-to-property relationships. Single-molecule break junction measurements yield a large number of conductance-distance traces, which are inherently highly stochastic. Here we propose a weakly supervised deep learning algorithm to classify and segment these conductance traces, a method that is mainly based on transfer learning with the pretrain-finetune technique. By exploiting the powerful feature extraction capabilities of the pretrained VGG-16 network, our convolutional neural network model not only achieves high accuracy in the classification of the conductance traces, but also segments precisely the conductance plateau from an entire trace with very few manually labeled traces. Thus, we can produce a more reliable estimation of the junction conductance and quantify the junction stability. These findings show that our model has achieved a better accuracy-to-manpower efficiency balance, opening up the possibility of using weakly supervised deep learning approaches in the studies of single-molecule junctions.

A de novo GRIN1 Variant Associated With Myoclonus and Developmental Delay: From Molecular Mechanism to Rescue Pharmacology.

N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.

High-Resolution Measurement of Molecular Internal Polarization Structure by Photoinduced Force Microscopy

Near-field interactions between metallic surfaces and single molecules play an essential role in the application of metamaterials. To reveal the near-field around a photo-irradiated single molecule on the metallic surface, high-resolution photo-assisted scanning microscopy is required. In this study, we theoretically propose photoinduced force microscopy (PiFM) measurements of single molecules at the atomic resolution. For experimental demonstration, we performed a numerical calculation of PiFM images of various transition states, including optical forbidden transitions, and interpreted them in terms of the interaction between the molecular internal polarization structures and localized plasmon. We also clarified the critical role of atomic-scale structures on the tip surface for high-resolution PiFM measurements.

Single Molecules Are Your Quanta: A Bottom-Up Approach toward Multidimensional Super-resolution Microscopy.

The rise of single-molecule localization microscopy (SMLM) and related super-resolution methods over the past 15 years has revolutionized how we study biological and materials systems. In this Perspective, we reflect on the underlying philosophy of how diffraction-unlimited pictures containing rich spatial and functional information may gradually emerge through the local accumulation of single-molecule measurements. Starting with the basic concepts, we analyze the uniqueness of and opportunities in building up the final picture one molecule at a time. After brief introductions to the more established multicolor and three-dimensional measurements, we highlight emerging efforts to extend SMLM to new dimensions and functionalities as fluorescence polarization, emission spectra, and molecular motions, and discuss rising opportunities and future directions. With single molecules as our quanta, the bottom-up accumulation approach provides a powerful conduit for multidimensional microscopy at the nanoscale.

Probing DNA-protein interactions using single-molecule diffusivity contrast

Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here, we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an anti-Brownian electrokinetic trap. We then cross-validate our method with protein-induced fluorescence enhancement, a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with protein-induced fluorescence enhancement allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.

Flexible, charged biopolymers in monovalent and mixed-valence salt: Regimes of anomalous electrostatic stiffening and of salt insensitivity.

The conformations of biological polyelectrolytes (PEs), such as polysaccharides, proteins, and nucleic acids, affect how they behave and interact with other biomolecules. Relative to neutral polymers, PEs in solution are more locally rigid due to intrachain electrostatic repulsion, the magnitude of which depends on the concentration of added salt. This is typically quantified using the Odijk-Skolnick-Fixman (OSF) electrostatic-stiffening model, in which salt-dependent Debye-Hückel (DH) screening modulates intrachain repulsion. However, the applicability of this approach to flexible PEs has long been questioned. To investigate this, we use high-precision single-molecule elasticity measurements to infer the scaling with salt of the local stiffness of three flexible biopolymers (hyaluronic acid, single-stranded RNA, and single-stranded DNA) in both monovalent and mixed-valence salt solutions. In monovalent salt, we collapse the data across all three polymers by accounting for charge spacing, and find a common power-law scaling of the electrostatic persistence length with ionic strength with an exponent of 0.66±0.02. This result rules out simple OSF pictures of electrostatic stiffening. It is roughly compatible with a modified OSF picture developed by Netz and Orland; alternatively, we posit the exponent can be explained if the relevant electrostatic screening length is the interion spacing rather than the DH length. In mixed salt solutions, we find a regime where adding monovalent salt, in the presence of multivalent salt, does not affect PE stiffness. Using coarse-grained simulations, and a three-state model of condensed, chain-proximate, and bulk ions, we attribute this regime to a "jacket" of ions surrounding the PE that regulates the chain's effective charge density as ionic strength varies. The size of this jacket in simulations is again consistent with a screening length controlled by interion spacing rather than the DH length. Taken together, our results describe a unified picture of the electrostatic stiffness of polyelectrolytes in the mixed-valence salt conditions of direct relevance to cellular and intercellular biological systems.

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerization and interaction with ArlI, the motor ATPase of the archaellum.

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Since autophosphorylation and dephosphorylation of KaiC are central properties for the function of KaiC, we asked whether autophosphorylation is also a property of ArlH proteins. We observed that both ArlH from the euryarchaeon Pyrococcus furiosus (PfArlH) and from the crenarchaeon Sulfolobus acidocaldarius (SaArlH) have autophosphorylation activity. Using a combination of single-molecule fluorescence measurements and biochemical assays, we show that autophosphorylation of ArlH is closely linked to its oligomeric state when bound to hexameric ArlI. These experiments also strongly suggest that ArlH is a hexamer in its ArlI-bound state. Mutagenesis of the putative catalytic residue (Glu-57 in SaArlH) in ArlH results in a reduced autophosphorylation activity and abolished archaellation and motility in S. acidocaldarius, indicating that optimum phosphorylation activity of ArlH is essential for archaellation and motility.

Role of sequence and position of the cleavage sites in prothrombin activation

In the penultimate step of the coagulation cascade, the multidomain vitamin-K-dependent zymogen prothrombin is converted to thrombin by the prothrombinase complex composed of factor Xa, cofactor Va, and phospholipids. Activation of prothrombin requires cleavage at two residues, R271 and R320, along two possible pathways generating either the intermediate prethrombin-2 (following initial cleavage at R271) or meizothrombin (following initial cleavage at R320). The former pathway is preferred in the absence of and the latter in the presence of cofactor Va. Several mechanisms have been proposed to explain this preference, but the role of the sequence and position of the sites of cleavage has not been thoroughly investigated. In this study, we engineered constructs where the sequences 261DEDSDRAIEGRTATSEYQT279 and 310RELLESYIDGRIVEGSDAE328 were swapped between the R271 and R320 sites. We found that in the absence of cofactor Va, the wild-type sequence at the R271 site is cleaved preferentially regardless of its position at the R271 or R320 site, whereas in the presence of cofactor Va, the R320 site is cleaved preferentially regardless of its sequence. Additional single-molecule FRET measurements revealed that the environment of R271 changes significantly upon cleavage at R320 due to the conformational transition from the closed form of prothrombin to the open form of meizothrombin. Detailed kinetics of cleavage at the R271 site were monitored by a newly developed assay based on loss of FRET. These findings show how sequence and position of the cleavage sites at R271 and R320 dictate the preferred pathway of prothrombin activation.

Water Splitting: Water Splitting Induced by Visible Light at a Copper‐Based Single‐Molecule Junction (Small 28/2021)

In article number 2008109, Marius Buerkle, Satoshi Kaneko, Tomoaki Nishino, and co-workers induce the water-splitting reaction under visible light utilizing a copper-based single-molecule junction. A product (hydrogen) and a reactant (water-molecule) are distinguished by the single-molecule measurements techniques based on conductance measurement, inelastic electron tunneling spectroscopy, and the first-principles calculations.

Different translation dynamics of β- and γ-actin regulates cell migration.

β- and γ-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and mesenchymal cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequences; however, it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in β- and γ-cytoplasmic actins' intracellular functions, using stable polyclonal populations of immortalized mouse embryonic fibroblasts with exogenously expressed actin isoforms and their 'codon-switched' variants. When targeted to the cell periphery using β-actin 3'UTR; β-actin and γ-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single-molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in β- and γ-actins' translation elongation rates in cells, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

Kindlin-3 disrupts an intersubunit association in the integrin LFA1 to trigger positive feedback activation by Rap1 and talin1.

Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLβ2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the β2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the β2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/β clasp) and unbending of the ectodomains of the α/β heterodimer. These conformational changes promoted high-affinity talin1 binding to the β2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.