Combining optical trapping, fluorescence microscopy and micro-fluidics for single molecule studies of DNA-protein interactions

Complexity and heterogeneity are common denominators of the many molecular events taking place inside the cell. Single-molecule techniques are important tools to quantify the actions of biomolecules. Heterogeneous interactions between multiple proteins, however, are difficult to study with these technologies. One solution is to integrate optical trapping with micro-fluidics and single-molecule fluorescence microscopy. This combination opens the possibility to study heterogeneous/complex protein interactions with unprecedented levels of precision and control. It is particularly powerful for the study of DNA-protein interactions as it allows manipulating the DNA while at the same time, individual proteins binding to it can be visualized. In this work, we aim to illustrate several published and unpublished key results employing the combination of fluorescence microscopy and optical tweezers. Examples are recent studies of the structural properties of DNA and DNA-protein complexes, the molecular mechanisms of nucleo-protein filament assembly on DNA and the motion of DNA-bound proteins. In addition, we present new results demonstrating that single, fluorescently labeled proteins bound to individual, optically trapped DNA molecules can already be tracked with localization accuracy in the sub-10 nm range at tensions above 1 pN. These experiments by us and others demonstrate the enormous potential of this combination of single-molecule techniques for the investigation of complex DNA-protein interactions.     

单分子水平高分子相互作用的AFM成像与单分子力谱研究

结合作者近期的研究工作,重点介绍了如何把原子力显微镜(AFM)成像及单分子力谱结合(包括原位结合或者离位结合)起来,研究高分子之间的相互作用.本文涉及生物高分子(主要是核酸-蛋白质体系)以及合成高分子体系(如聚氧乙烯,PEO)的相关研究工作.对于生物高分子体系,主要以长链核酸(如双螺旋DNA及RNA)为探针,首先利用A...     

Quantitative Assessment of Tip Effects in Single‐Molecule High‐Speed Atomic Force Microscopy Using DNA Origami Substrates

High‐speed atomic force microscopy (HS‐AFM) is widely employed in the investigation of dynamic biomolecular processes at a single‐molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami‐based single‐molecule assay. Despite its femtomolar dissociation constant, we find that HS‐AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami‐based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS‐AFM imaging.     

DNA curtains and nanoscale curtain rods: high-throughput tools for single molecule imaging

Single molecule visualization of protein-DNA complexes can reveal details of reaction mechanisms and macromolecular dynamics inaccessible to traditional biochemical assays. However, these techniques are often limited by the inherent difficulty of collecting statistically relevant information from experiments explicitly designed to look at single events. New approaches that increase throughput capacity of single molecule methods have the potential for making these techniques more readily applicable to a variety of biological questions involving different types of DNA transactions. Here we show that nanofabricated chromium barriers, which are located at strategic positions on a fused silica slide otherwise coated with a supported lipid bilayer, can be used to organize DNA molecules into molecular curtains. The DNA that makes up the curtains is visualized by total internal reflection fluorescence microscopy (TIRFM) allowing simultaneous imaging of hundreds or thousands of aligned molecules. These DNA curtains present a robust experimental platform portending massively parallel data acquisition of individual protein-DNA interactions in real time.     

Programmable Assembly of DNA-protein Hybrid Structures

DNA is the genetic information carrier for most known living organisms on Earth,while proteins are the functional component that carry out most biological processes.Many natural machineries are DNA-protein hybrid complexes to cooperatively and efficiently conduct sophisticated biological tasks.It has drawn increasing interest to the research field to construct artificial DNA-protein hybrid structures towards a variety of applications including biological studies,nanofabrication,biomedical research,etc.In this regard,here in this report we reviewed the up-to-date progress on making DNA-protein hybrid structures,with a particular focus on DNA nanotechnology-enabled programmable assembly of DNA-protein hybrid structures.     

Complexes of cationic block copolymer micelles with DNA: histone/DNA complex mimetics

The formation of complexes between cationic polymeric micelles of PS-b-PQ2VP amphiphilic block copolymers and DNA molecules in aqueous solutions is investigated at pH = 7. The physicochemical characteristics of the "polyplexes" at different DNA/polymer ratios were characterized in terms of mass, size and charge using static, dynamic and electrophoretic light scattering and AFM. The complexes are spherical and assume their maximum size and mass around the charge stoichiometric ratio. After addition of increased amounts of salt in the solutions, partial dissociation of the systems was observed. The present systems can be considered as mimetics of histone/DNA complexes formed under physiological conditions in living cells.     

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.     

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.     

DNA imaged on a HOPG electrode surface by AFM with controlled potential

Single-molecule AFM imaging of single-stranded and double-stranded DNA molecules self-assembled from solution onto a HOPG electrode surface is reported. The interaction of DNA with the hydrophobic surface induced DNA aggregation, overlapping, intra- and intermolecular interactions. Controlling the electrode potential and using the phase images as a control method, to confirm the correct topographical characterization, offers the possibility to enlarge the capability of AFM imaging of DNA immobilized onto conducting substrates, such as HOPG. The application of a potential of +300 mV (versus AgQRE) to the HOPG enhanced the robustness and stability of the adsorbed DNA molecules, increasing the electrostatic interaction between the positively charged electrode surface and the negatively charged DNA sugar-phosphate backbone.     

Single-molecule studies on DNA and RNA.

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

Assembly of DNA Nanostructures with Branched Tris‐DNA

Branched tris‐DNA, in which two oligonucleotides of the same sequence and one other oligonucleotide of a different sequence are connected with a rigid central linker, was prepared chemically by using a DNA synthesizer. Two branched tris‐DNA molecules with complementary DNA sequences form dimer and tetramer as well as linear and spherical oligomer complexes. The complex formation was studied by UV/thermal denaturation, enzyme digestion, gel electrophoresis, and AFM imaging.     

Quantitative Assessment of Tip Effects in Single-Molecule High-Speed Atomic Force Microscopy Using DNA Origami Substrates

High-speed atomic force microscopy (HS-AFM) is widely employed in the investigation of dynamic biomolecular processes at a single-molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami-based single-molecule assay. Despite its femtomolar dissociation constant, we find that HS-AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami-based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS-AFM imaging.     

铬(VI)-谷胱甘肽配合物诱导小牛胸腺DNA变性的新表征方法

本文采用电化学方法对铬(VI)-谷胱甘肽(GSH)配合物诱导DNA变性进行表征，同时运用原子力显微镜(AFM)对DNA损伤变性过程进行可视化探测。结果表明：在pH=5.6的HAc-NaAc缓冲溶液中，DNA溶液中加入Cr(VI)-GSH配合物后进行循环伏安扫描，+0.20 V和0.00 V(vs SCE)处出现一对新的氧化还原峰，该氧化还原峰随Cr(VI)-GSH配合物浓度增加峰电流上升。DNA热变性和表面活性剂SDS变性实验进一步证明了该峰为DNA变性后的氧化还原峰，且变性DNA的峰信号在修饰电极上比裸金电极上更为灵敏。电化学动力学表明在30 min内配合物诱导DNA变性的程度随时间的上升而增加，并通过AFM观察了配合物作用下DNA断链的过程。     

Electrospray‐Ionization Mass Spectrometry for Screening the Specificity and Stability of Single‐Stranded‐DNA Templated Self‐Assemblies

Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.     

DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.     

Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay

Native gel electrophoresis (mobility shift) assays may be used to obtain quantitative information about the site distribution, equilibria and kinetics of protein-DNA interactions. These applications depend on the ability of the electrophoretic system to resolve the reaction components, and on their stabilities during the separation process. Factors which affect the lifetimes and mobilities of protein-DNA complexes during electrophoresis include reaction and electrophoresis buffer composition, pH, and ionic strength; the presence of low molecular weight effectors and enzymatic substrates; the nature and concentration of the gel matrix; the temperature; the molecular weights of protein and DNA; the stoichiometric ratios of their complexes; and the possibility of conformational and configurational isomerization of reaction components. We discuss how these factors influence the acquisition of quantitative data from electrophoretic patterns and band intensities, and present formulas for the estimation of equilibrium constants and rate constants for prototypical DNA-protein interactions.     

Hybridization with nanostructures of single-stranded DNA

Nanostructures of single-stranded DNA (ssDNA) were produced within alkanethiol self-assembled monolayers using nanografting, an atomic force microscopy (AFM) based lithography technique. Next, variations of the fabrication parameters, such as the concentration of ssDNA or lines per frame, allowed for the regulation of the density of ssDNA molecules within the nanostructures. The label-free hybridization of nanostructures, monitored using high-resolution AFM imaging, has proven to be highly selective and sensitive; as few as 50 molecules can be detected. The efficiency of the hybridization reaction at the nanometer scale highly depends on the ssDNA packing density within the nanostructures. This investigation provides a fundamental step toward sensitive DNA detection and construction of complex DNA architectures on surfaces.     

Studies of DNA-protein interactions by gel electrophoresis

The use of gel electrophoresis in studies of nucleic acid-protein (especially DNA-protein) interactions has yielded much qualitative and quantitative information about a variety of such systems. The reduction in mobility of complexes relative to free DNA allows isolation and characterization of the complexes as well as determination of thermodynamic and kinetic properties of the interactions. This article begins with a review of recent applications of the "gel retardation" assay, by way of introduction to experiments in two areas. In the first, a hypothesis is tested regarding whether a DNA molecule with sizable proteins bound very near to each end migrates through a polyacrylamide gel differently than does the corresponding complex having the proteins in the middle of the DNA fragment. The data show little mobility differences for these types of complexes, implying that both may move in a linear, "snakelike", manner through the gel. The experiments also provide results pertaining to questions of DNA bending caused by the binding of the E. coli catabolite activator protein (CAP) and RNA polymerase to the lactose promoter region. It appears that DNA bending by CAP at its wild type lac binding site is retained in complexes where RNA polymerase is bound simultaneously at the lac UV5 promoter.     

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions

DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically-interlocked minicircle rotaxane and catenane inside a frame-shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA-protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.     

Effects of the biological backbone on stacking interactions at DNA-protein interfaces: the interplay between the backbone···π and π···π components

The (gas-phase) MP2/6-31G*(0.25) π···π stacking interactions between the five natural bases and the aromatic amino acids calculated using (truncated) monomers composed of conjugated rings and/or (extended) monomers containing the biological backbone (either the protein backbone or deoxyribose sugar) were previously compared. Although preliminary energetic results indicated that the protein backbone strengthens, while the deoxyribose sugar either strengthens or weakens, the interaction calculated using truncated models, the reasons for these effects were unknown. The present work explains these observations by dissecting the interaction energy of the extended complexes into individual backbone···π and π···π components. Our calculations reveal that the total interaction energy of the extended complex can be predicted as a sum of the backbone···π and π···π components, which indicates that the biological backbone does not significantly affect the ring system through π-polarization. Instead, we find that the backbone can indirectly affect the magnitude of the π···π contribution by changing the relative ring orientations in extended dimers compared with truncated dimers. Furthermore, the strengths of the individual backbone···π contributions are determined to be significant (up to 18 kJ mol(-1)). Therefore, the origin of the energetic change upon model extension is found to result from a balance between an additional (attractive) backbone···π component and differences in the strength of the π···π interaction. In addition, to understand the effects of the biological backbone on the stacking interactions at DNA-protein interfaces in nature, we analyzed the stacking interactions found in select DNA-protein crystal structures, and verified that an additive approach can be used to examine the strength of these interactions in biological complexes. Interestingly, although the presence of attractive backbone···π contacts is qualitatively confirmed using the quantum theory of atoms in molecules (QTAIM), QTAIM electron density analysis is unable to quantitatively predict the additive relationship of these interactions. Most importantly, this work reveals that both the backbone···π and π···π components must be carefully considered to accurately determine the overall stability of DNA-protein assemblies.