DNA mechanics as a tool to probe helicase and translocase activity

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

Analysis of kinetics in noisy systems: application to single molecule tethered particle motion

In the tethered particle motion method the length of a DNA molecule is monitored by measuring the range of diffusion of a microsphere tethered to the surface of a microscope coverslip through the DNA molecule itself. Looping of DNA (induced by binding of a specific protein) can be detected with this method and the kinetics of the looping/unlooping processes can be measured at the single molecule level. The microsphere's position variance represents the experimental variable reporting on the polymer length. Therefore, data windowing is required to obtain position variance from raw position data. Due to the characteristic diffusion time of the microsphere, the low-pass filtering required to attain a good signal/noise ratio (S/N) in the discrimination of looped versus unlooped state impacts significantly the measurement's time resolution. Here we present a method for measuring lifetimes based on half-amplitude thresholding and then correcting the kinetic measurements, taking into account low S/N (leading to false events) and limited time resolution (leading to missed events). This method allows an accurate and unbiased estimation of the kinetic parameters under investigation, independently of the choice of the window used for variance calculation, with potential applications to other single molecule measurements with low S/N.     

MELAS和MERRF综合征相关mtDNA突变位点检测集成芯片的建立

摘要: 文中建立了一种新型的寡核苷酸芯片, 用于线粒体脑肌病伴高乳酸血症和卒中样发作(Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes, MELAS)和肌阵挛性癫痫伴发不规整红纤维(Myoclonic epilepsy with ragged red fibers, MERRF)线粒体DNA所有已知突变位点的集成检测。将31对allele位点特异性的寡核苷酸探针包被在醛基修饰的载玻片表面, 以多重不对称PCR方法制备Cy5荧光标记靶基因。利用此芯片对5例MELAS患者、5例MERRF患者及20例健康对照进行筛查, 结果发现, MELAS患者均为MT-T1基因A3243G突变; 在MERRF患者组, MT-TK基因A8344G突变4例, T8356C突变1例; 健康对照组均未发现31种相关mtDNA突变。芯片检测与DNA测序结果完全一致。结果表明, 这种寡核苷酸芯片可以对MELAS和MERRF综合征已知突变位点进行同步快速检测, 具有较高的灵敏度和特异性。这一模式的基因芯片经过适当改装后也可用于其他人类线粒体疾病的基因诊断。     

Detection of biological macromolecules on a biochip dedicated to UV specific absorption

This work describes an ultraviolet biosensing technique based on specific molecular absorption detected with a previously developed spectrally selective aluminum gallium nitride (AlGaN) based detector. Light absorption signal of DNA and proteins, respectively at 260 nm and 280 nm, is used to image biochips. To allow detection of protein or DNA monolayers at the surface of a biochip, we develop contrast-enhancing multilayer substrates. We analyze them through models and experiments and validate the possibility of measuring absorptions of the order of 10(-3). These multilayer structures display a high reflectivity, and maximize the interaction of the electric field with the biological element at the chip surface. Optimization of the experimental absorption, which includes effects such as roughness of the biochip, spectral and angular resolution of the optics, illumination, etc., is carried out with an inorganic ultraviolet absorber (titanium dioxide) deposit. We obtained an induced absorption contrast enhanced by a factor of 4.0, conferring enough sensitivity to detect monolayers of DNA or proteins. Experimental results on an Escherichia coli histidine-tagged methionyl-tRNA synthetase protein before and after complexation with an anti-polyHis specific antibody validate our biosensing technique. This label-free optical method may be helpful in controlling biochip coatings, and subsequent biological coupling at the surface of a biochip.     

Single molecule techniques in DNA repair: A primer

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.     

Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids.     

Microsecond single-molecule tracking (μsSMT)

Here we report on a method to track individual molecules on nanometer length and microsecond timescales using an optical microscope. Our method is based on double-labeling of a molecule with two spectrally distinct fluorophores and illuminating it with laser pulses of different wavelengths that partially overlap temporally. We demonstrate our method by using it to resolve the motion of short DNA oligomers in solution down to a timescale of 100 μs.     

Single-particle tracking for DNA tether length monitoring

We describe a simple single-particle tracking approach for monitoring the length of DNA molecules in tethered particle motion experiments. In this method, the trajectory of a submicroscopic bead tethered by a DNA molecule to a glass surface is determined by videomicroscopy coupled to image analysis. The amplitude of motion of the bead is measured by the standard deviation of the distribution of successive positions of the bead in a given time interval. We were able to describe theoretically the variation of the equilibrium value of the amplitude of the bead motion with the DNA tether length for the entire applicable DNA length range (up to ~3500 bp). The sensitivity of the approach was illustrated by the evidence obtained for conformational changes introduced into a Holliday junction by the binding of the Escherichia coli RuvA protein. An advantage of this method is that the trajectory of the tethered bead, rather than its averaged motion, is measured, allowing analysis of the conformational dynamics of DNA chains at the single-molecule level.     

Probing DNA conformational changes with high temporal resolution by tethered particle motion

The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.     

Microsecond Single-Molecule Tracking (μsSMT)

Here we report on a method to track individual molecules on nanometer length and microsecond timescales using an optical microscope. Our method is based on double-labeling of a molecule with two spectrally distinct fluorophores and illuminating it with laser pulses of different wavelengths that partially overlap temporally. We demonstrate our method by using it to resolve the motion of short DNA oligomers in solution down to a timescale of 100 μs.     

Cohort analysis of a single nucleotide polymorphism on DNA chips

A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.     

生物芯片技术

生物芯片技术近年来发展极为迅速。生物芯片这一概念出现在２０世纪８０年代初,９０年代以来随着人类基因组计划研究的深入,生物芯片技术也得以飞速发展。本文将对生物芯片的概念、发展做一全面的叙述,并详细地介绍最新的生物芯片,如ＤＮＡ芯片等的基本原理、分类、制备,以及生物芯片的发展动向和应用前景。     

Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection

We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to approximately 10 nm was shown by scanning immobilized 100 nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody-receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

Single-molecule DNA digestion by lambda-exonuclease.

We used a bead displacement sensor to determine the enzymatic shortening of individual molecules of unstained lambda-DNA attached to optically trapped beads. The setup has been described previously (Dapprich and Nicklaus: Bioimaging 6:25-32, 1998) and works by observing the change in position of a trapped bead depending on its viscous drag force during motion. The drag force of a naked bead increases with each attached DNA molecule to a characteristic level that depends on the length and the number of DNAs per bead. A single undigested DNA molecule on a bead will remain stable for extended periods and exhibit a constant drag force in flow. If lambda-exonuclease is added, the drag force decreases from the level for one strand of DNA on a bead to that of a naked bead in about 45 min. This result indicates that the digestion of native lambda-DNA by lambda-exonuclease occurs at an average rate of approximately 15-20 Hz.