Parallel arrays of geometric nanowells for assembling curtains of DNA with controlled lateral dispersion

The analysis of individual molecules is evolving into an important tool for biological research, and presents conceptually new ways of approaching experimental design strategies. However, more robust methods are required if these technologies are to be made broadly available to the biological research community. To help achieve this goal we have combined nanofabrication techniques with single-molecule optical microscopy for assembling and visualizing curtains comprised of thousands of individual DNA molecules organized at engineered diffusion barriers on a lipid bilayer-coated surface. Here we present an important extension of this technology that implements geometric barrier patterns comprised of thousands of nanoscale wells that can be loaded with single molecules of DNA. We show that these geometric nanowells can be used to precisely control the lateral distribution of the individual DNA molecules within curtains assembled along the edges of the engineered barrier patterns. The individual molecules making up the DNA curtain can be separated from one another by a user-defined distance dictated by the dimensions of the nanowells. We demonstrate the broader utility of these patterned DNA curtains in a novel, real time restriction assay that we refer to as dynamic optical restriction mapping, which can be used to rapidly identify entire sets of cleavage sites within a large DNA molecule.     

单分子检测进展

单分子检测做为针对有限可数的化学微观个体性质和行为的测量分析方法,能够提供用传统的宏观测量方法得不到的分子微环境中微观个体信息,受到广泛关注。本文综述了近年来单分子检测领域的进展和单分子检测的基本技术,重点介绍了用电化学方法进行单分子检测的发展状况,并展望了单分子检测的发展前景。     

Yoctoliter Thermometry for Single‐Molecule Investigations: A Generic Bead‐on‐a‐Tip Temperature‐Control Module

A new temperature‐jump (T‐jump) strategy avoids photo‐damage of individual molecules by focusing a low‐intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T‐jump of 65 °C within milliseconds. To measure the temperature in situ in single‐molecule experiments, the temperature‐dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead‐on‐a‐tip module and the robust single‐molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T‐jump and single‐molecule techniques.     

Nonspecificity induces chiral specificity in the folding transition of giant DNA

The all-or-none type DNA folding transition from a coil to globule can be differentiated by the chirality of the triamines. The fluorescent microscope observation on single DNA molecules makes it clear that the tripeptides obtained from naturally occurring basic amino acids (l-lysine or l-arginine) can compact DNA molecules at concentrations lower than those from d-isomers. Nanometer-sized beads are found in the AFM images on the folded DNA molecule.     

How to accelerate protein search on DNA: location and dissociation

One of the most important features of biological systems that controls their functioning is the ability of protein molecules to find and recognize quickly specific target sites on DNA. Although these phenomena have been studied extensively, detailed mechanisms of protein-DNA interactions during the search are still not well understood. Experiments suggest that proteins typically find their targets fast by combining three-dimensional and one-dimensional motions, and most of the searching time proteins are non-specifically bound to DNA. However these observations are surprising since proteins diffuse very slowly on DNA, and it seems that the observed fast search cannot be achieved under these conditions for single proteins. Here we propose two simple mechanisms that might explain some of these controversial observations. Using first-passage time analysis, it is shown explicitly that the search can be accelerated by changing the location of the target and by effectively irreversible dissociations of proteins. Our theoretical predictions are supported by Monte Carlo computer simulations.     

Indirect micromanipulation of single molecules in water-in-oil emulsion

Based on real-time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time. Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques. We have developed a chemical reaction system that uses water droplets in oil as containers of materials. The water droplets can be manipulated by optical force. The manipulation of the water droplets permits the fusion of two selected droplets. This process corresponds to mixing of different samples. We designate this system as "w/o (water-in-oil emulsion) microreactor system", and each droplet can be thought of as a "microreactor". In this system, single molecules can be manipulated readily, as a molecule can be contained in a microm-sized microreactor. The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short. The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA. This fusion induced a rapid binding of YOYO.     

A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single-pulse UV Laser Irradiation

Abstract— Photocrosslinking of proteins to DNA by single-pulse UV laser has been used only in analytical experiments, carried out with reconstituted complexes of a single DNA binding protein and a labeled target sequence. Here we propose a large-scale technique for irradiation of nuclei, generating preparative quantities of covalently linked protein-DNA complexes for further analysis of the partner molecules. The use of a flow cuvette allows a milligram of DNA in either nuclei or chromatin to be irradiated by a single pulse within few minutes. The efficiency of crosslinking varies from 6 to 12% of the total nuclear proteins. The presence of histones and other chromosomal proteins in the crosslinked protein-DNA complexes was demonstrated by using specific antibodies. The irradiation procedure can be fully automated using a microcomputer.     

Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy

A novel approach is presented for studying the kinetics of specific protein-DNA interactions by NMR exchange spectroscopy. The experimental design involves the direct observation of translocation of a homeodomain between cognate sites on two oligonucleotide duplexes, differing by only a single base pair at the edge of the DNA recognition sequence. The single base-pair change perturbs the 1H-15N correlation spectrum of a number of residues, while leaving the affinity for the DNA unchanged. The exchange process has apparent rate constants in the 5-20 s-1 range which are linearly dependent upon the concentration of free DNA. These rates are about 3 orders of magnitude larger than the dissociation rate constant determined by gel shift assays at nanomolar DNA concentrations. The complete NMR exchange data set, comprising auto- and cross-peak intensities as a function of mixing time at five concentrations of free DNA, can be fit simultaneously to a simple model in which protein translocation between DNA duplexes occurs via a second-order process (with rate constants of approximately 6 x 104 M-1 s-1) involving direct collision of a protein-DNA complex with free DNA. This is akin to intersegmental transfer, and a physical model for the process is discussed. Rapid translocation at high concentrations of free DNA observed directly by NMR exchange spectroscopy reconciles the long half-lives of protein-DNA complexes measured by biochemical analysis in vitro with the highly dynamic behavior of such complexes observed in vivo. The relevance of this mechanism to the kinetics of protein-DNA interactions within the cell is discussed.     

A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions

The application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the thermodynamic analysis of protein-DNA complexes is described. A series of five model protein-DNA complexes involving two known DNA binding proteins, Arc repressor and CopG, were analyzed in order to determine the accuracy, precision, and generality of the SUPREX technique for quantifying the strength of protein-DNA interactions. For protein-DNA complexes that reversibly unfold in a two-state manner, we demonstrate that reasonably precise Kd values in agreement with those determined by conventional techniques can be determined by SUPREX. In the case of protein-DNA complexes that are not well modeled by a two-state unfolding mechanism, we find that relative binding affinities can be determined in the SUPREX experiment.     

Molecular beacons: a novel DNA probe for nucleic acid and protein studies

A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.     

Nonspecific protein-DNA interactions: complexation of alpha-chymotrypsin with a genomic DNA

In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the F?rster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.     

Non-equilibrium capillary electrophoresis of equilibrium mixtures--appreciation of kinetics in capillary electrophoresis

We describe a new electrophoretic method (patent pending), Non-Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM), and demonstrate its application to the study of protein-DNA interactions. A single NECEEM experiment allows for the determination of equilibrium and kinetic parameters of protein-DNA complex formation. The equilibrium mixture is prepared by mixing protein and DNA; it contains three components: free protein, free DNA, and the protein-DNA complex. A small plug of such a mixture is injected onto a capillary and the three components are separated under non-equilibrium conditions using a run buffer that does not contain the components of the equilibrium mixture. The protein-DNA complex decays during the NECEEM separation; the resulting electropherograms contain characteristic peaks and exponential curves. A simple analysis of a single electropherogram reveals two parameters: the equilibrium dissociation constant of the protein-DNA complex and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the ratio of the two experimentally-determined constants. NECEEM was applied to find the equilibrium and kinetic parameters of interaction between an E. coli single-stranded DNA binding protein and a fluorescently-labeled oligonucleotide. The constants determined by NECEEM are in good agreement with those obtained by other methods. The new method is simple, fast, and accurate. It can be equally applied to other non-covalent molecular complexes.     

Conformation and luminescence of isolated molecular semiconductor molecules

In this article, we describe, for the first time, direct comparisons of the detailed structures of two small molecule organic semiconductors, oligo(phenylenvinylene) (OPV) molecules with chains of five and six phenyl rings (5R-OC(8)H(17) and 6R-OC(8)H(17)), respectively, and their luminescence properties on a single molecule level. Our data originate from a combination of two powerful diagnostic tools in physical chemistry: ion mobility and single molecule fluorescence spectroscopy. These techniques enable us to precisely determine the shapes of isolated molecules in the gas phase and to correlate these structures to the emission from single molecules supported on bare glass substrates. The principal structural uncertainty in OPVs is the (possible) presence and location of cis-vinylene linkages (cis-defects) in the oligomer. The results show that the structures observed in the gas phase are strongly correlated to the categories of molecules observed in the single molecule polarization anisotropy measurements with nearly identical distributions for the two OPV molecules studied. Each category is also characterized by the luminescence efficiency of the molecules in each class, providing a direct correlation between the luminescence efficiency and the shape of the molecule. This combination of techniques provides a level of information far beyond that obtained via any other analytical technique.     

Visualization of large elongated DNA molecules

Long and linear DNA molecules are the mainstream single‐molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA‐protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.     

Kinetics of Photoinduced Reactions at the Single-Molecule Level: The KACB Method

The kinetics of photoinduced reactions can be approached by laser flash photolysis techniques. Although such techniques allow for a detailed understanding of the important photophysics of molecules, they normally require a substantial amount of sample for measurements (>1 nmol), and thus, they are difficult to apply to analytical and diagnostic applications. The photophysics of a fluorescent molecule can be accessed by monitoring the kinetics of the fluctuation of fluorescence, which is called blinking. Blinking is a phenomenon that can be monitored only if molecules are observed at the single-molecule level. In bulk solution, blinking kinetics can be measured by using fluorescence correlation spectroscopy (FCS), which normally requires more than 10⁵ times less sample than that required for laser flash photolysis. Blinking is controlled to extract fruitful microenvironmental information around a fluorescent molecule, by using a method named kinetic analysis based on the control of fluorescence blinking (KACB). This Concept highlights the adaption of the KACB method to investigate the local conformation of DNA with less than 1 pmol of DNA sample.     

Self-assembly of irregular graphs whose edges are DNA helix axes

A variety of computational models have been introduced recently that are based on the properties of DNA. In particular, branched junction molecules and graphlike DNA structures have been proposed as computational devices, although such models have yet to be confirmed experimentally. DNA branched junction molecules have been used previously to form graph-like three-dimensional DNA structures, such as a cube and a truncated octahedron, but these DNA constructs represent regular graphs, where the connectivities of all of the vertexes are the same. Here, we demonstrate the construction of an irregular DNA graph structure by a single step of self-assembly. A graph made of five vertexes and eight edges was chosen for this experiment. DNA branched junction molecules represent the vertexes, and duplex molecules represent the edges; in contrast to previous work, specific edge molecules are included as components. We demonstrate that the product is a closed cyclic single-stranded molecule that corresponds to a double cover of the graph and that the DNA double helix axes represent the designed graph. The correct assembly of the target molecule has been demonstrated unambiguously by restriction analysis.     

Base pair distance analysis in single DNA molecule by direct stochastic optical reconstruction microscopy

Precise fluorescence imaging of single λ-DNA molecules for base pair distance analysis requires a superresolution technique, as these distances are on the order of diffraction limit. Individual λ-DNA molecules intercalated with the fluorescent dye YOYO-1 were investigated at subdiffraction spatial resolution by direct stochastic optical reconstructionmicroscopy (dSTORM). Various dye-to-DNA base pair ratios were imaged by photoswitching YOYO-1 between the fluorescent state and the dark state using two laser sources. The acquired images were reconstructed into a super-resolution image by applying Gaussian fitting to the centroid of the point spread function. By measuring the distances between localized fluorophores, the base pair distances in single DNA molecules for dye-to-DNA base pair ratios of 1:50, 1:100, and 1:500 were calculated to be 17.1±0.8 nm, 34.3±2.2 nm, and 170.3±8.1 nm, respectively, which were in agreement with theoretical values. These results demonstrate that intercalating dye in a single DNA molecule can be photoswitched without the use of an activator fluorophore, and that super-localization precision at a spatial resolution of ~17 nm was experimentally achieved.     

DNA sequencing with titanium nitride electrodes

We construct a hydrogen‐bond based metal–molecule–metal junction, which contains two identical “reader” molecules, one single DNA base as a bridged molecule, and two titanium nitride electrodes. Hydrogen bonds are formed between “reader” molecules and DNA base, whereas titanium–sulfur bonds are formed between “reader” molecules and titanium nitride electrodes. We perform electronic structure calculations for both the bare bridged molecule and the full metal–molecule–metal system. The projected density of states shows that when the molecule is connected to the titanium nitride electrode, the energy levels of the bridged molecule are shifted, with an indirect effect on the hydrogen bonds. This is similar to the case for a gold electrode but with a more pronounced effect. We also calculate the current–voltage characteristics for the molecular junctions containing each DNA base. Results show that titanium nitride as an electrode can generate distinct conductance for each DNA base, providing an alternative electrode for DNA sequencing. © 2013 Wiley Periodicals, Inc.     

An integrated optics microfluidic device for detecting single DNA molecules

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.