Stretched DNA Molecules Research Articles

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is theCDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Correlated Single-Molecule Magnetic Tweezers and Fluorescence Measurements of DNA-Enzyme Interactions.

Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.

Resonator nanophotonic standing-wave array trap for single-molecule manipulation and measurement

Nanophotonic tweezers represent emerging platforms with significant potential for parallel manipulation and measurements of single biological molecules on-chip. However, trapping force generation represents a substantial obstacle for their broader utility. Here, we present a resonator nanophotonic standing-wave array trap (resonator-nSWAT) that demonstrates significant force enhancement. This platform integrates a critically-coupled resonator design to the nSWAT and incorporates a novel trap reset scheme. The nSWAT can now perform standard single-molecule experiments, including stretching DNA molecules to measure their force-extension relations, unzipping DNA molecules, and disrupting and mapping protein-DNA interactions. These experiments have realized trapping forces on the order of 20 pN while demonstrating base-pair resolution with measurements performed on multiple molecules in parallel. Thus, the resonator-nSWAT platform now meets the benchmarks of a table-top precision optical trapping instrument in terms of force generation and resolution. This represents the first demonstration of a nanophotonic platform for such single-molecule experiments.

Direct unfolding of RuvA-HJ complex at the single-molecule level

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ∼2-fold. Compared with the ΔGunfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging.

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System

Conformational transitions from secondary (e.g., B- to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several single-molecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil-globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

Effects of AC/DC Electric Fields on Stretching DNA Molecules

The effects of AC/DC electric fields on stretching DNA molecules were discussed in this work. In the experiments of stretching DNA molecules with AC/DC electric fields, the voltage range was changed from 0[Formula: see text]V to 10[Formula: see text]V, and the frequency of AC electric field was kept at 50[Formula: see text]kHz. An atomic force microscope (AFM) was used to obtain DNA distributions under different electric fields. DNA molecules were curved and randomly distributed in solution if there was not any force applied to them. When an AC electric field was applied to the DNA sample, the curvature of DNA molecules was decreased gradually, and the stretching result was more obvious with the increase of voltage from 0.1[Formula: see text]V to 5[Formula: see text]V. The DNA molecules were broken when the voltage was increased to 6[Formula: see text]V. However, under the DC electric field, the stretching result of DNA molecules reached to their optimum state when the voltage was 2[Formula: see text]V, and they kept their steady state even though larger electric field intensities applied to the electrodes. The results can be used for the study of DNA–DNA, protein–DNA and quantum dot–DNA interactions and for the exploration of DNA biophysical properties.

Optimization of nonlinear optical tweezers suitable to stretch DNA molecules without broken state

The positioning of the trapped bead and DNA’s stretching dynamics in the nonlinear optical tweezers are numerically simulated by finite difference method using general Langevin. From the performance of longitudinal position-pulling time curves of bead and evolution of forces controlled by average laser power, the trapping time, possible maximum stretched length, and laser intensity threshold of to broken DNA molecule are found. We also discussed the suitable conditions to avoid broken and overstretched states for longitudinally optical stretched DNA molecule. Finally, the optimized configuration of NOT for DNA molecules having different contour lengths is proposed.

An experimental investigation of attraction between knots in a stretched DNA molecule

It has been predicted through simulations that two knots in a stretched polymer will feel an attraction towards one another and will tend to be found in close proximity. Here, we examine data from experiments in which we stretched knotted DNA molecules and investigate a subset which contained two or more knots. In contrast to the expected behavior of an isolated knot in an elongational field, we observe that pairs of knots move towards each other from distant sections of the molecule. After meeting, pairs of knots fluctuate in and out of visual contact, and can remain in proximity for minutes at a time. Our experimental results suggest that knots on an extended polymer experience an attractive interaction.

Nonlinear microscope objective using thin layer of organic dye for optical tweezers

The microscope objective for optical tweezers requires a high resolving power and a large numerical aperture. For these purposes, the oil immersion objective can be improved. However, the index of the oil immersion objective and glass cover-slip must be matched resulting in a limit to enhance numerical aperture, and then optical trap efficiency. The optical trap efficiency can be enhanced by using a thin layer of organic dyelike an added nonlinear lens. To improve the resolving power and the optical trap efficiency of nonlinear tweezers, a thin layer of organic dye glued by microscope objective like the nonlinear microscope objective is proposed in this paper. The explicit expressions of the focal length, numerical aperture, axial optical trap efficiency of nonlinear microscope objective are theoretically derived. Some interesting properties of particular nonlinear microscope objective used like nonlinear optical tweezersare numerically observed. The obtained results show that the numerical aperture and optical trap efficiency are significantly improved. Our proposed nonlinear microscope objective has advantages such as more compact and smooth focal shift in the range of micrometers suitable to stretch DNA molecules in comparison with the previous ones.

Real-Time Condensation of Nanoconfined DNA by an Intrinsically Disordered Polycationic Protein

The Hepatitis C viral core protein (HCVcp) is an intrinsically disordered nucleocapsid protein that induces structural transformations in the viral RNA necessary for its efficient packaging within the viral particle. The mechanism of the nucleoprotein complex (NPC) formation is mainly driven by electrostatic interactions, where HCVcp locally screens repulsion charges by acting as a macromolecular counterion (+22). In the later stage of viral assembly, the number of HCVcp molecules increases above a critical threshold concentration after which all NPCs are transformed into nucleocapsid like particles (NLP). Herein, we have used single DNA molecule nanofluidics in combination with fluorescence microscopy to monitor the interaction between HCVcp and double stranded DNA. In our novel dynamic nanofluidic device, we can confine single DNA molecules in the nanochannels and visualize their condensation under constant inflow of proteins. We observed that NPC formation incur changes in the mechanical properties of stretched DNA molecules. These changes are primarily marked by appearance of a local compaction at either end of the stretched DNA, which serves as a nucleation site for global compaction. The length of the stretched DNA molecules gradually decreases and reaches a condensed state at above threshold concentrations of HCVcp. We were also able to follow unpacking of the condensed state in real time. Proteinase K was introduced from another inlet of the dynamic device and it gradually removed the HCVcp from DNA, which in turn lead to an increase in extension of condensed DNA. Our data allowed us to investigate the formation and dissociation of the DNA:HCVcp complex in real time. On a broader level, our results highlight the use of nanofluidic channels for studying complex and dynamic DNA-protein interactions.

High-Performance Image-Based Measurements of Biological Forces and Interactions in a Dual Optical Trap

Abstract Optical traps enable nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, precise and fast image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection, however successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive. Here we develop a camera-based detection platform that enables exceptionally accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision, a performance level previously only achieved using photodiodes. We then use the DNA unzipping technique to localize bound proteins with extraordinary sub-base-pair precision, revealing how thermal DNA fluctuations allow an unzipping fork to sense and respond to a bound protein prior to a direct encounter. This work significantly advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

High-Performance Image-Based Measurements of Biological Forces and Interactions in a Dual Optical Trap.

Optical traps enable the nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection; however, successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive. Here, we develop a camera-based detection platform that enables accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision. We then use the DNA unzipping technique to localize bound proteins with sub-base-pair precision, revealing how thermal DNA "breathing" fluctuations allow an unzipping fork to detect and respond to the presence of a protein bound downstream. This work advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

Transverse dielectrophoretic-based DNA nanoscale confinement

Confinement of single molecules within nanoscale environments is crucial in a range of fields, including biomedicine, genomics, and biophysics. Here, we present a method that can concentrate, confine, and linearly stretch DNA molecules within a single optical field of view using dielectrophoretic (DEP) force. The method can convert an open surface into one confining DNA molecules without a requirement for bonding, hydrodynamic or mechanical components. We use a transverse DEP field between a top coverslip and a bottom substrate, both of which are coated with a transparent conductive material. Both layers are attached using double-sided tape, defining the chamber. The nanofeatures lie at the “floor” and do not require any bonding. With the application of an alternating (AC) electric field (2 Vp-p) between the top and bottom electrodes, a DEP field gradient is established and used to concentrate, confine and linearly extend DNA in nanogrooves as small as 100-nm in width. We also demonstrate reversible loading/unloading of DNA molecules into nanogrooves and nanopits by switching frequency (between 10 kHz to 100 kHz). The technology presented in this paper provides a new method for single-molecule trapping and analysis.

Efficient DNA-assisted synthesis of trans-membrane gold nanowires

Whereas electric circuits and surface-based (bio)chemical sensors are mostly constructed in-plane due to ease of manufacturing, 3D microscale and nanoscale structures allow denser integration of electronic components and improved mass transport of the analyte to (bio)chemical sensor surfaces. This work reports the first out-of-plane metallic nanowire formation based on stretching of DNA through a porous membrane. We use rolling circle amplification (RCA) to generate long single-stranded DNA concatemers with one end anchored to the surface. The DNA strands are stretched through the pores in the membrane during liquid removal by forced convection. Because the liquid–air interface movement across the membrane occurs in every pore, DNA stretching across the membrane is highly efficient. The stretched DNA molecules are transformed into trans-membrane gold nanowires through gold nanoparticle hybridization and gold enhancement chemistry. A 50 fM oligonucleotide concentration, a value two orders of magnitude lower than previously reported for flat surface-based nanowire formation, was sufficient for nanowire formation. We observed nanowires in up to 2.7% of the membrane pores, leading to an across-membrane electrical conductivity reduction from open circuit to <20 Ω. The simple electrical read-out offers a high signal-to-noise ratio and can also be extended for use as a biosensor due to the high specificity and scope for multiplexing offered by RCA.

Force determination in lateral magnetic tweezers combined with TIRF microscopy.

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.

Electrostatic confinement and manipulation of DNA molecules for genome analysis

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte "issues" as exploitable advantages by our invention and characterization of the "molecular gate," which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an "electrostatic bottle." This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasmaflorum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

Twisting short dsDNA with applied tension

The twisting deformation of mechanically stretched DNA molecules is studied by a coarse grained Hamiltonian model incorporating the fundamental interactions that stabilize the double helix and accounting for the radial and angular base pair fluctuations. The latter are all the more important at short length scales in which DNA fragments maintain an intrinsic flexibility. The presented computational method simulates a broad ensemble of possible molecule conformations characterized by a specific average twist and determines the energetically most convenient helical twist by free energy minimization. As this is done for any external load, the method yields the characteristic twist-stretch profile of the molecule and also computes the changes in the macroscopic helix parameters i.e. average diameter and rise distance. It is predicted that short molecules under stretching should first over-twist and then untwist by increasing the external load. Moreover, applying a constant load and simulating a torsional strain which over-twists the helix, it is found that the average helix diameter shrinks while the molecule elongates, in agreement with the experimental trend observed in kilo-base long sequences. The quantitative relation between percent relative elongation and superhelical density at fixed load is derived. The proposed theoretical model and computational method offer a general approach to characterize specific DNA fragments and predict their macroscopic elastic response as a function of the effective potential parameters of the mesoscopic Hamiltonian.

Influence of concentration on distribution properties of stretched-DNA in the MEC studied with fluorescence imaging and drop shape analyzing

Stretching and manipulating DNA efficiently is significant for exploring the properties and applications of single DNA molecules. Here, the influence of concentrations of buffer and DNA on properties of stretched DNA molecules in the molecular evaporation combing (MEC) is investigated systematically with the single molecule fluorescence imaging microscopy and the high-precision drop shape analyzing technology. The stretched degree and uniformity of combed DNA molecules decrease as the buffer concentration are increased from 7 to 20mM. When the buffer concentration changes from 12 to 15mM, the stretched DNA molecules are apt to form a ringlike pattern. During the MEC process, there exist two kinds of evaporation modes, i.e., the constant contact angle mode and the constant contact radius mode. The former only takes effect in the lower concentration of buffer and DNA, enabling the uniform stretching. While the latter plays the leading role in the higher concentration, promoting the formation of the ringlike pattern of DNA molecules.

Determination of Electrophoretic Forces in a Gel Matrix in Different Ionic Strength Conditions

Nanocoding, a genome analysis platform, relies on very low ionic strength conditions to elongate DNA molecules up to 1.06 (fully stretched DNA molecule = 1)1, which is the largest stretch reported in the literature. Understanding how electroosmotic and electrophoretic forces vary, as ionic strength decreases, will enable better Nanocoding devices to be developed. Using two different gel assays to determine electroosmotic and overall mobility (includes contributions from electrophoretic forces and electroosmotic forces), we were able to determine electrophoretic mobility in different ionic strength solutions. Our first gel assay relied on electrophoresising linear DNA molecules [pUC19 (2.7 kb), pBR322 (4.4 kb), φX174 (5.4 kb), and PSNAPf-H2B (6.2 kb)] in varying gel concentrations (1.5%, 1.25%, 1%, 0.75%, 0.5%) to determine the free solution mobility (overall mobility) of these molecules in dilutions of TE buffer. As the buffer concentration decreases from 2X (Ionic strength = 13.8 mM) to 1X (Ionic strength = 7.3 mM), the overall mobility increased. As we further diluted TE (< 1X TE), the overall mobility drastically decreased as the ionic strength decreased.1. Kounovsky-Shafer, K.L., Hernandez-Ortiz, J.P., Jo, K., Odijk, T., de Pablo, J.J., and David C. Schwartz, D.C. Presentation of large DNA molecules for analysis as nanoconfined dumbbells, Macromolecules, 2013, 46 (20), 8356-8368.