DNA Unzipping Research Articles

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.

Possible scenarios of DNA double-helix unzipping process in single-molecule manipulation experiments.

Single-molecule experiments on DNA unzipping are analyzed on the basis of the mobility of nucleic bases in complementary pairs. Two possible scenarios of DNA double-helix unzipping are proposed and studied, using the atom-atom potential function method. According to the first scenario, the base pairs transit into a 'preopened' metastable state and then fully open along the 'stretch' pathway. In this case, the DNA unzipping takes place slowly and as an equilibrium process, with the opening energies being similar to the energies obtained in thermodynamic experiments on DNA melting. The second scenario is characterized by higher opening forces. In this case, the DNA base pairs open directly along the 'stretch' pathway. It follows from our calculations that, in this scenario, the enthalpy difference between the A[Formula: see text]T and G[Formula: see text]C base pairs is much higher than in the first case. The features of the first unzipping scenario show that it can play a key role during the process of DNA genetic information transfer in vivo. It follows from our study that a peculiarity of the second scenario is that it can be used for the development of faster methods for reading genetic information in vitro.

Conjugated Polymer/Graphene Oxide Complexes for Photothermal Activation of DNA Unzipping and Binding to Protein

DNA–protein interactions control DNA transcription, recombination, restriction, and replication, which play an important role in regulating life activities. We developed a new strategy to photothermally regulate DNA unzipping and binding to single-stranded binding protein (SSBP) based on the hybrid systems of graphene oxide (GO) and conjugated polymer. GO is applied as the photothermal modulator to activate the unzipping of dsDNA into ssDNA. Upon near-infrared (NIR) laser irradiation, the thermal melting transition of DNA is promoted, which results in much farther distance between poly[(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP) and fluorescein labeled at the terminus of the DNA in comparison to that of dsDNA before NIR irradiation in the presence of GO. Therefore, the fluorescence resonance energy transfer (FRET) efficiency for PFP/fluorescein becomes weaker upon irradiation. Moreover, FRET efficiency could readily recover when the unzipped DNA hybridizes with SSBP....

Single-Molecule DNA Unzipping Reveals Asymmetric Modulation of the Transcription Factor EGR-1 by its Binding Site Sequence and Context

Binding of transcription factors to regulatory elements is a central step in the complex regulation of gene expression, and its perturbation is linked to numerous disease states. Egr-1 is an inducible transcription factor that binds to 9-bp response elements via three zinc finger domains in response to a variety of stimuli, such as hormonal signals and stress. In our work, we use single-molecule DNA unzipping with optical tweezers to study the binding properties of Egr-1 to its binding sites, using as a model the promoter of the Lhb gene. We find that both the core 9 base pairs bound to Egr-1 in each of the binding sites on Lhb, and the base pairs flanking these sites, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking the triplet in contact with zinc finger 3. Next, using a novel method to characterize the dissociation time of Egr-1 at the single molecule level, we show that a local, mechanical perturbation of the interactions of zinc finger 3 is able to destabilize the complex more effectively than a similar perturbation acting on the interactions of ZF1. Taken together, our findings suggest a novel functional role for ZF3 in the interaction of Egr-1 with other proteins.

DNA unzipping with asymmetric periodic forces: Robustness of the scaling behavior of hysteresis loop

We study the effect of periodic unzipping forces (symmetric and asymmetric) on the steady-state hysteresis loop area of force-extension curves of DNA. For the triangular force, we get back the previously reported scaling exponents but for the ratchet force, we find that the scaling exponents deviate from the reported ones. We also study the temperature dependence of the scaling exponents for the triangular force. At the low-frequency regime, the choice of the scaling form determines whether the scaling exponents depend on the temperature or not.

Measuring Unzipping and Rezipping of Single Long DNA Molecules with Optical Tweezers.

The unwinding of double-stranded DNA is a frequently occurring event during the cellular processes of DNA replication, repair, and transcription. To help further investigate properties of this fundamental process as well as to study proteins acting on unzipped DNA at the single molecule level, we describe a novel method for efficient preparation of long DNA constructs (arbitrary sequences of many kilobasepairs (kbp) in length) that can be forcibly unzipped and manipulated with optical tweezers or other single-molecule manipulation techniques. This method utilizes PCR, a nicking endonuclease, and strand displacement synthesis by the Klenow fragment of DNA polymerase I to introduce labeled nucleotides at appropriate positions to facilitate unzipping of the DNA by application of force. We also describe various optical tweezers measurement modes for measuring DNA unzipping and rezipping. These methods have applications to studying helicases and DNA binding proteins.

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context.

Most functional transcription factor (TF) binding sites deviate from their ‘consensus’ recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used single-molecule DNA unzipping with optical tweezers to study how Egr-1, a TF harboring three zinc fingers (ZF1, ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 bp bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein–DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

Class 2 CRISPR-Cas RNA-guided endonucleases: Swiss Army knives of genome editing.

CRISPR-Cas is a bacterial defense system against phage infection and nucleic acid invasion. Class 2 type II CRISPR-Cas9 has also been widely used for genome engineering. Here, we review novel insights into the CRISPR class 2 type V enzymes, specifically Cpf1 and C2c1, which display different DNA-recognition and cleavage characteristics than those of Cas9, the best-characterized member of class 2. Recent structures of these ribonucleoprotein complexes that capture several stages of the endonuclease reaction have provided molecular details of recognition, unzipping and cleavage of the target DNA, allowing their comparison with Cas9. A detailed understanding of these mechanisms is crucial for improving these genome engineering tools and expanding the genomic space that can be targeted.

Single molecule high-throughput footprinting of small and large DNA ligands

Most DNA processes are governed by molecular interactions that take place in a sequence-specific manner. Determining the sequence selectivity of DNA ligands is still a challenge, particularly for small drugs where labeling or sequencing methods do not perform well. Here, we present a fast and accurate method based on parallelized single molecule magnetic tweezers to detect the sequence selectivity and characterize the thermodynamics and kinetics of binding in a single assay. Mechanical manipulation of DNA hairpins with an engineered sequence is used to detect ligand binding as blocking events during DNA unzipping, allowing determination of ligand selectivity both for small drugs and large proteins with nearly base-pair resolution in an unbiased fashion. The assay allows investigation of subtle details such as the effect of flanking sequences or binding cooperativity. Unzipping assays on hairpin substrates with an optimized flat free energy landscape containing all binding motifs allows determination of the ligand mechanical footprint, recognition site, and binding orientation.

The Structure of Short and Genomic DNA at the Interparticle Junctions of Cationic Nanoparticles

Current understanding of the mechanisms underlying noncovalent interactions between native DNA and nanoparticles, as well as their impact on the double‐helix structure, is far from providing a comprehensive view. It is known that these interactions are largely defined by the physicochemical properties of the metal/liquid interface, in particular by the nanoparticle surface charge. Remarkably, while DNA unzipping upon binding with cationic nanoparticles is reported, the exact determinants of this structural perturbation remain unclear. Herein, plasmon‐based spectroscopies (surface‐enhanced Raman scattering (SERS) and surface‐plasmon resonance (SPR) and theoretical simulations are combined to directly investigate the role of the cooperative binding of cationic nanoparticles with different surface charges on the structural integrity of a large variety of DNAs. The intrinsic nature of the SERS effect unlocks the possibility of selectively examining the impact of nanoparticle clustering on the duplex structure over a wide degree of colloidal aggregation and without the need of external intercalating dyes or strand labeling. This extensive work provides new fundamental insights into the interaction between nucleic acids and nanoparticles, addressing key questions regarding the role played by multiple variables such as the nanoparticle surface charge, the DNA‐mediated cluster size and geometry, and nucleic acids' length, composition, and conformational properties.

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage.

Cpf1 is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we provide insight into its DNA-targeting mechanism by determining the structure of Francisella novicida Cpf1 with the triple-stranded R-loop generated after DNA cleavage. The structure reveals the machinery involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner into the double-stranded DNA. Unzipping of the double-stranded DNA occurs in a cleft arranged by acidic and hydrophobic residues facilitating the crRNA-DNA hybrid formation. The PAM single-stranded DNA is funnelled towards the nuclease site through a mixed hydrophobic and basic cavity. In this catalytic conformation, the PAM-interacting domain and the helix-loop-helix motif in the REC1 domain adopt a 'rail' shape and 'flap-on' conformations, respectively, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms the 'septum', separating the displaced PAM strand and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1.

Multiplexed Force Spectroscopy using DNA Nanoswitch a Centrifuge

We present high-throughput single-molecule manipulation using a benchtop centrifuge, overcoming limitations common in other single-molecule approaches such as cost, throughput, technical difficulty, and infrastructure requirements. An inexpensive and compact Centrifuge Force Microscope adapts to a commercial centrifuge enabling use by non-specialists, and integration of DNA nanoswitches facilitates reliable measurements. We demonstrate new features by measuring temperature and magnesium dependent DNA unzipping together with multiplexed DNA force-extension and overstretching.

Characterization of Interstrand DNA-DNA Cross-Links Using the α-Hemolysin Protein Nanopore.

Nanopore-based sensors have been studied extensively as potential tools for DNA sequencing, characterization of epigenetic modifications such as 5-methylcytosine, and detection of microRNA biomarkers. In the studies described here, the α-hemolysin protein nanopore embedded in a lipid bilayer was used for the detection and characterization of interstrand cross-links in duplex DNA. Interstrand cross-links are important lesions in medicinal chemistry and toxicology because they prevent the strand separation that is required for read-out of genetic information from DNA in cells. In addition, interstrand cross-links are used for the stabilization of duplex DNA in structural biology and materials science. Cross-linked DNA fragments produced unmistakable current signatures in the nanopore experiment. Some cross-linked substrates gave irreversible current blocks of >10 min, while others produced long current blocks (10-100 s) before the double-stranded DNA cross-link translocated through the α-hemolysin channel in a voltage-driven manner. The duration of the current block for the different cross-linked substrates examined here may be dictated by the stability of the duplex region left in the vestibule of the nanopore following partial unzipping of the cross-linked DNA. Construction of calibration curves measuring the frequency of cross-link blocking events (1/τon) as a function of cross-link concentration enabled quantitative determination of the amounts of cross-linked DNA present in samples. The unique current signatures generated by cross-linked DNA in the α-HL nanopore may enable the detection and characterization of DNA cross-links that are important in toxicology, medicine, and materials science.

Diffusive dynamics of DNA unzipping in a nanopore.

When an electric field is applied to an insulating membrane, movement of charged particles through a nanopore is induced. The measured ionic current reports on biomolecules passing through the nanopore. In this work, we explored the kinetics of DNA unzipping in a nanopore using our coarse-grained model (Stachiewicz and Molski, J. Comput. Chem. 2015, 36, 947). Coarse graining allowed a more detailed analysis for a wider range of parameters than all-atom simulations. Dependence of the translocation mode (unzipping or distortion) on the pore diameter was examined, and the threshold voltages were estimated. We determined the potential of mean force, position-dependent diffusion coefficient, and position-dependent effective charge for the DNA unzipping. The three molecular profiles were correlated with the ionic current and molecular events. On the unzipping/translocation force profile, two energy maxima were found, one of them corresponding to the unzipping, and the other to the translocation barriers. The unzipping kinetics were further explored using Brownian dynamics.

Physical origin of DNA unzipping.

In DNA transcription, the base pairs are unzipped in response to the enzymatic forces, separating apart two intertwined nucleotide strands. Consequently, the double-stranded DNA (dsDNA), in which two nucleotide strands wind about each other, transits structurally to the single-stranded DNA (ssDNA) in which two nucleotide strands are completely unwound and separated. The large interstrand separation is intimately related to the softening nucleotide strands. This conceptual framework is reinforced with the flow of the bending modulus toward zero under recursion relations derived from the momentum shell renormalization group. Interestingly, the stretch modulus remains the same under recursion relations. The renormalization of the bending modulus to zero has a profound implication that ssDNA has the shorter bending persistence length than does dsDNA in accordance with experiments.

A Temperature-Jump Optical Trap for Single-Molecule Manipulation

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability. Our design has features to reduce convection and baseline shifts, which have troubled previous heating-laser instruments. As proof of accuracy, we used the instrument to carry out DNA unzipping experiments in which we derived the average basepair free energy, entropy, and enthalpy of formation of the DNA duplex in a range of temperatures between 5°C and 50°C. We also used the instrument to characterize the temperature-dependent elasticity of single-stranded DNA (ssDNA), where we find a significant condensation plateau at low force and low temperature. Oddly, the persistence length of ssDNA measured at high force seems to increase with temperature, contrary to simple entropic models.

Thermal and inertial resonances in DNA unzipping.

Single-molecule experiments combined with alternate forces are able to provide useful information not present in standard constant-force and -velocity pulling protocols. Here, we study the effects of such forces in the DNA mechanical unzipping by using an extension of the Peyrard-Bishop-Dauxois model. By changing the damping regime in the dynamical equations, we obtained two resonant mechanisms in both the mean time and the mean force of unzipping. One is thermally assisted and it is characterized by a matching between the period of the external force and the mean unzipping time of the DNA chain, while the other depends on the inertial properties of the system. Both mechanisms are studied systematically under different opening protocols and different parameters of the system. The main results here presented contribute in characterizing and finding optimized conditions in DNA unzipping experiments.

Free-Energy Landscape and Characteristic Forces for the Initiation of DNA Unzipping

DNA unzipping, the separation of its double helix into single strands, is crucial in modulating a host of genetic processes. Although the large-scale separation of double-stranded DNA has been studied with a variety of theoretical and experimental techniques, the minute details of the very first steps of unzipping are still unclear. Here, we use atomistic molecular-dynamics simulations, coarse-grained simulations, and a statistical-mechanical model to study the initiation of DNA unzipping by an external force. Calculation of the potential of mean force profiles for the initial separation of the first few terminal basepairs in a DNA oligomer revealed that forces ranging between 130 and 230 pN are needed to disrupt the first basepair, and these values are an order of magnitude larger than those needed to disrupt basepairs in partially unzipped DNA. The force peak has an echo of ∼50 pN at the distance that unzips the second basepair. We show that the high peak needed to initiate unzipping derives from a free-energy basin that is distinct from the basins of subsequent basepairs because of entropic contributions, and we highlight the microscopic origin of the peak. To our knowledge, our results suggest a new window of exploration for single-molecule experiments.

单分子拉伸力实验中DNA解链动力学无序效应的介观统计研究

Recent single molecule pulling experiments on DNA unzipping have revealed a heavy-tailed rupture force distribution, indicating dynamic disorder. Here we present a theoretical framework based on a generalized Langevin equation (GLE) with fractional Gaussian noise (fGn) and power-law memory kernel to study the kinetics of DNA unzipping to address the effects of dynamic disorder on barrier-crossing kinetics under external pulling force. Such a GLE-fGn strategy is associated with the slow conformational fluctuations of macromolecules. By using the Kramers rate theory, we have obtained analytical formulae for the time-dependent rate coefficient k ( t , F ), and the distribution of rupture force p ( F ) which demonstrates a fat tail at high force as functions of time t and force F . In addition, through fitting our results to data from single molecule pulling experiments on DNA unzipping, we estimate the kinetic parameters and qualify the extent of disorder. Our work provides a plausible interpretation for the evidence of dynamic disorder in force-induced DNA unzipping, compared to two other models based on Bell model and Langevin equation (LE) approach.