Gaining insight into the nanoscale properties of sol-gel-derived silicate thin films by single-molecule spectroscopy

The application of single-molecule spectroscopic methods in studies of individual nanoscale environments within sol-gel-derived silicate thin films is reviewed. Representative examples of the experiments performed and results obtained in several studies from the authors' laboratories are given. Included are investigations of the static and dynamic polarity properties of organically modified silicate (ORMOSIL) films. The results of these studies point to nonrandom variations in the film properties, providing strong evidence for the formation of phase-separated organic- and inorganic-rich domains. Studies of single-molecule diffusion through the same films yield important evidence for the formation of liquidlike silicate oligomers that facilitate probe molecule diffusion. Finally, single-molecule studies of the local pH within individual film environments are discussed. Valuable information on the contributions of local materials' acidity variations to overall sample heterogeneity is obtained. The results of immersion studies indicate that certain molecular environments are inaccessible to external solutions over periods as long as a few hours. The article concludes with a discussion of possible future challenges in this research that may be addressed by new and existing single-molecule methods.     

Accounting for observed small angle X‐ray scattering profile in the protein–protein docking server cluspro

The protein‐protein docking server ClusPro is used by thousands of laboratories, and models built by the server have been reported in over 300 publications. Although the structures generated by the docking include near‐native ones for many proteins, selecting the best model is difficult due to the uncertainty in scoring. Small angle X‐ray scattering (SAXS) is an experimental technique for obtaining low resolution structural information in solution. While not sufficient on its own to uniquely predict complex structures, accounting for SAXS data improves the ranking of models and facilitates the identification of the most accurate structure. Although SAXS profiles are currently available only for a small number of complexes, due to its simplicity the method is becoming increasingly popular. Since combining docking with SAXS experiments will provide a viable strategy for fairly high‐throughput determination of protein complex structures, the option of using SAXS restraints is added to the ClusPro server. © 2015 Wiley Periodicals, Inc.     

Go-and-Back method: effective estimation of the hidden motion of proteins from single-molecule time series

We present an effective method for estimating the motion of proteins from the motion of attached probe particles in single-molecule experiments. The framework naturally incorporates Langevin dynamics to compute the most probable trajectory of the protein. By using a perturbation expansion technique, we achieve computational costs more than 3 orders of magnitude smaller than the conventional gradient descent method without loss of simplicity in the computation algorithm. We present illustrative applications of the method using simple models of single-molecule experiments and confirm that the proposed method yields reasonable and stable estimates of the hidden motion in a highly efficient manner.     

Dwell time analysis of a single-molecule mechanochemical reaction

Force-clamp spectroscopy is a novel technique for studying mechanochemistry at the single-bond level. Single disulfide bond reduction events are accurately detected as stepwise increases in the length of polyproteins that contain disulfide bonds and that are stretched at a constant force with the cantilever of an atomic force microscope (AFM). The kinetics of this reaction has been measured from single-exponential fits to ensemble averages of the reduction events. However, exponential fits are notoriously ambiguous to use in cases of kinetic data showing multiple reaction pathways. Here we introduce a dwell time analysis technique, of widespread use in the single ion channel field, that we apply to the examination of the kinetics of reduction of disulfide bonds measured from single-molecule force-clamp spectroscopy traces. In this technique, exponentially distributed dwell time data is plotted as a histogram with a logarithmic time scale and a square root ordinate. The advantage of logarithmic histograms is that exponentially distributed dwell times appear as well-defined peaks in the distribution, greatly enhancing our ability to detect multiple kinetic pathways. We apply this technique to examine the distribution of dwell times of 4488 single disulfide bond reduction events measured in the presence of two very different kinds of reducing agents: tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) and the enzyme thioredoxin (TRX). A different clamping force is used for each reducing agent to obtain distributions of dwell times on a similar time scale. In the case of TCEP, the logarithmic histogram of dwell times showed a single peak, corresponding to a single reaction mechanism. By contrast, similar experiments done with TRX showed two well-separated peaks, marking two distinct modes of chemical reduction operating simultaneously. These experiments demonstrate that dwell time analysis techniques are a powerful approach to studying chemical reactions at the single-molecule level.     

Novel fluorophores for single-molecule imaging

Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.     

Single-molecule studies on DNA and RNA.

DNA and RNA are the most individual molecules known. Therefore, single-molecule experiments with these nucleic acids are particularly useful. This review reports on recent experiments with single DNA and RNA molecules. First, techniques for their preparation and handling are summarised including the use of AFM nanotips and optical or magnetic tweezers. As important detection techniques, conventional and near-field microscopy as well as fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) are touched on briefly. The use of single-molecule techniques currently includes force measurements in stretched nucleic acids and in their complexes with binding partners, particularly proteins, and the analysis of DNA by restriction mapping, fragment sizing and single-molecule hybridisation. Also, the reactions of RNA polymerases and enzymes involved in DNA replication and repair are dealt with in some detail, followed by a discussion of the transport of individual nucleic acid molecules during the readout and use of genetic information and during the infection of cells by viruses. The final sections show how the enormous addressability in nucleic acid molecules can be exploited to construct a single-molecule field-effect transistor and a walking single-molecule robot, and how individual DNA molecules can be used to assemble a single-molecule DNA computer.     

Single-molecule force spectroscopy of selenium-containing amphiphilic block copolymer: toward disassembling the polymer micelles

Selenium-containing polymers are a new type of responsive polymer material. Here, a selenium-containing amphiphilic block copolymer (PEG-PUSe-PEG) has been investigated using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). The deviation between force-extension curves of PEG-PUSe-PEG in water and in DMSO is found to be related to the disassembly of the micellar structures in water. SMFS experiments on PEG-PUSeox-PEG suggest that the change from selenide to oxidized selenone contributes significantly to the change in amphiphilicity, without obviously influencing the single-chain elasticity.     

Elementary processes of DNA surface hybridization resolved by single-molecule kinetics: implication for macroscopic device performance

Direct monitoring of single-molecule reactions has recently become a promising means of mechanistic investigation. However, the resolution of reaction pathways from single-molecule experiments remains elusive, primarily because of interference from extraneous processes such as bulk diffusion. Herein, we report a single-molecule kinetic investigation of DNA hybridization on a metal surface, as an example of a bimolecular association reaction. The tip of the scanning tunneling microscope (STM) was functionalized with single-stranded DNA (ssDNA), and hybridization with its complementary strand on an Au(111) surface was detected by the increase in the electrical conductance associated with the electron transport through the resulting DNA duplex. Kinetic analyses of the conductance changes successfully resolved the elementary processes, which involve not only the ssDNA strands and their duplex but also partially hybridized intermediate strands, and we found an increase in the hybridization efficiency with increasing the concentration of DNA in contrast to the knowledge obtained previously by conventional ensemble measurements. The rate constants derived from our single-molecule studies provide a rational explanation of these findings, such as the suppression of DNA melting on surfaces with higher DNA coverage. The present methodology, which relies on intermolecular conductance measurements, can be extended to a range of single-molecule reactions and to the exploration of novel chemical syntheses.

Hybridization of a single DNA molecule on a surface was investigated by electrical conductance measurements. The hybridization efficiency increases with increasing the DNA concentration, in contrast to preceding studies with ensemble studies.     

Force-reactivity property of a single monomer is sufficient to predict the micromechanical behavior of its polymer

We demonstrate an accurate prediction of the micromechanical behavior of a single chain of cyclopropanated polybutadiene, which is governed by rapid isomerization of the cyclopropane moieties at ~1.2 nN, from the force-rate correlation of this reaction measured in a small series of increasingly strained macrocycles. The data demonstrate that a single physical quantity, force, uniquely defines the dynamics across length scales from >100 to <1 nm and that strain imposed through molecular design and that imposed by micromanipulation techniques have equivalent effects on the kinetics of a chemical reaction. This represents a new method of screening potential monomers for applications in stress-responsive materials that could also facilitate atomistic interpretations of single-molecule force experiments.     

Pairwise interactions between linear alkanes in water measured by AFM force spectroscopy

Pairwise interactions between n-alkanes from decane to octadecane in water have been studied by single-molecule force spectroscopy. The interacting molecules are covalently tethered to the glass substrate and to the probe of an atomic force microscope by water-soluble linkers to facilitate single-molecule detection. However, the measured distribution of rupture forces deviates significantly from the distribution predicted by theoretical models for rupture of individual bonds. To describe the statistics of rupture forces, an analytical model that considers near-simultaneous rupture of two bonds loaded by tethers with different lengths is introduced. The common most probable force analysis approach is used for comparison. In both data analyses, the possible systematic errors due to nonlinear elasticity of polymeric tethers and variations in the shape of the potential of mean force were considered. Experimental distributions of rupture forces are well-fit by the two-bond rupture model using a single set of kinetic parameters for different experiments, while the most probable force approach yields parameters that vary significantly for different samples. The measured activation energies for dissociation of alkanes are close to the free energies predicted by cavity models of hydrophobic interactions. The surface free-energy density is estimated to be approximately 21 kJ/(mol nm (2)) and is close to the upper limit of free energies used in the computer simulations of hydrophobic interactions in proteins. In contrast to the predictions of the cavity models, the measured activation energy does not increase monotonically with increase in alkane chain size. To explain this discrepancy and the measured distance to the transition-state barrier (approximately 0.6 nm), it is suggested that alkanes undergo conformational transition to the collapsed state upon dimerization. Change in the alkane conformation from extended to helical has been observed previously for binding of alkanes in water to hydrophobic synthetic receptors. Here, however, conformational change is suggested without geometrical constraints imposed by small cavitands. The proposed collapsed state of the alkane dimers has implications for the kinetics of self-assembly of surfactant micelles.     

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.     

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.     

A new method for the covalent attachment of DNA to a surface for single-molecule studies

Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA-protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (> 60pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.     

原子力显微镜-荧光显微镜联用技术在活细胞单分子检测中的应用

原子力显微镜(Atomic force microscopy,AFM)及荧光显微镜(Fluorescence microscopy,FM)是目前活细胞单分子分析检测中最常用的两种工具.结合两种显微镜的优势,发展高时空分辨、多功能的AFM-FM联用技术成为近年该领域的研究热点.本文简述了AFM单分子力谱和FM单分子荧光成像的原理,总结了AFM-FM联用系统在仪器研制方面的发展概况,并结合本课题组在应用AFM-FM联用技术研究细胞膜上配受体相互作用等方面的工作,介绍了其在活细胞单分子检测中的应用进展.     

Mechanically activated molecular switch through single-molecule pulling

We investigate a prototypical single-molecule switch marrying force spectroscopy and molecular electronics far from the thermodynamic limit. We use molecular dynamics to simulate a conducting atomic force microscope mechanically manipulating a molecule bound to a surface between a folded state and an unfolded state while monitoring the conductance. Both the complexity and the unique phenomenology of single-molecule experiments are evident in this system. As the molecule unfolds/refolds, the average conductance reversibly changes over 3 orders of magnitude; however, throughout the simulation the transmission fluctuates considerably, illustrating the need for statistical sampling in these systems. We predict that emergent single-molecule signatures will still be evident with conductance blinking, correlated with force blinking, being observable in a region of dynamic bistability. Finally, we illustrate some of the structure-function relationships in this system, mapping the dominant interactions in the molecule for mediating charge transport throughout the pulling simulation.     

Single-Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions

The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron-sulfur protein containing a unique Fe₂S₂(His)₁(Cys)₃ metal cluster with a single Fe−N(His87) coordinating bond. This labile Fe−N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe−N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM-SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single-molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe−N bond.     

Single-molecule force-clamp spectroscopy: dwell time analysis and practical considerations

Single-molecule force-clamp spectroscopy has become a powerful tool for studying protein folding/unfolding, bond rupture, and enzymatic reactions. Different methods have been developed to analyze force-clamp spectroscopy data on polyproteins to obtain kinetic parameters characterizing the mechanical unfolding of proteins, which are often modeled as a two-state process (a Poisson process). However, because of the finite number of domains in polyproteins, the statistical analysis of the force-clamp spectroscopy data is different from that of a classical Poisson process, and the equivalency of different analysis methods remains to be proven. In this article, we show that these methods are equivalent and lead to accurate measurements of the unfolding rate constant. We also demonstrate that distinct from the constant-pulling-velocity experiments, in which the unfolding rate extracted from the data is dependent on the number of protein domains in the polyproteins (the N effect), force-clamp experiments do not show any N effect. Using a simulated data set, we also highlighted important practical considerations that one needs to take into account when using the single-molecule force-clamp spectroscopy technique to characterize the unfolding energy landscape of proteins.     

聚合物单分子力谱的研究进展

在单分子水平研究聚合物体系的分子内及分子间相互作用, 对于揭示其结构-性能的关系, 进而实现对相应功能的调控极为重要. 基于原子力显微镜技术(AFM)的单分子力谱, 由于其操作简单且适用面广, 在单分子研究领域得到了广泛的应用. 本文概括了该技术在生物高分子及合成高分子体系的研究进展. 对于生物高分子体系, 主要介绍了核酸(DNA/RNA)、 蛋白质和多糖(淀粉)的单分子力谱研究及利用各自力学指纹谱对其它分子间的相互作用的研究. 对于合成高分子体系, 主要介绍了聚合物的一级结构与单链弹性的关系及溶剂和聚集态结构等对高分子单链力学性质的影响规律.     

生物纳米孔道技术在非基因测序方面的研究与应用

纳米孔道分析技术是一种低成本、快速、无需标记的单分子检测技术,仅有20多年的发展历史,在DNA单分子测序领域展示出较好的应用前景,现已有商业化的产品面世且趋于成熟.越来越多的研究表明,纳米孔可作为一个通用的单分子传感器.本文综述了生物纳米孔道分析技术对蛋白质、多肽和核酸等单个分子与孔道间相互作用、动力学和热力学过程的实时监测以及多种生物大分子和金属离子的定量检测等方面的研究进展.在纳米孔技术中,电化学检测系统也十分重要,本文还特别介绍了高带宽及超低电流分辨仪器和相关软件的相关进展.