This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes.
Atomic force microscopy; AFM; mica functionalization; surface chemistry; silanes; silatranes; DNA structure and dynamics; protein-DNA complexes
The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.
APOBEC3G; single-stranded DNA binding proteins; site search mechanisms; atomic force microscopy; AFM; high-speed AFM
The APOBEC3 family of DNA cytosine deaminases functions to block the spread of endogenous retroelements and retroviruses including HIV-1. Potency varies among family members depending on the type of parasitic substrate. APOBEC3A (A3A) is unique among the human enzymes in that it is expressed predominantly in myeloid lineage cell types, it is strongly induced by innate immune agonists such as type 1 interferon, and it has the capacity to accommodate both normal and 5-methyl cytosine nucleobases. Here we apply atomic force microscopy (AFM) to characterize the interaction between A3A and single- and double-stranded DNA using a hybrid DNA approach in which a single-stranded region is flanked by defined length duplexes. AFM image analyses reveal A3A binding to single-stranded DNA, and that this interaction becomes most evident (∼80% complex yield) at high protein-to-DNA ratios (at least 100∶1). A3A is predominantly monomeric when bound to single-stranded DNA, and it is also monomeric in solution at concentrations as high as 50 nM. These properties agree well with recent, biochemical, biophysical, and structural studies. However, these characteristics contrast with those of the related enzyme APOBEC3G, which in similar assays can exist as a monomer but tends to form oligomers in a concentration-dependent manner. These AFM data indicate that A3A has intrinsic biophysical differences that distinguish it from APOBEC3G. The potential relationships between these properties and biological functions in innate immunity are discussed.
Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations and in a broad range of pH. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.
Atomic force microscopy; AFM; Mica functionalization; Surface chemistry; Silanes; Silatranes; DNA structure and dynamics; Protein-DNA complexes
Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that Escherichia coli SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg2+ cations or a high ionic strength. In the absence of Mg2+ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg2+ cations.
The DNA cytosine deaminase APOBEC3G (A3G) is capable of blocking retrovirus replication by editing viral cDNA and impairing reverse transcription. However, the biophysical details of this host-pathogen interaction are unclear. Here we applied atomic force microscopy (AFM) and hybrid DNA substrates to investigate properties of A3G bound to single-stranded DNA (ssDNA). Hybrid DNA substrates included ssDNA with 5′ or 3′ ends attached to DNA duplexes (tail-DNA) and gap-DNA substrates, in which ssDNA is flanked by two double-stranded fragments. We found that A3G binds with similar efficiency to the 5′ and 3′ substrates, suggesting that ssDNA polarity is not an important factor. Additionally, we observed that A3G binds the single-stranded region of the gap-DNA substrates with the same efficiency as tail-DNA. These results demonstrate that single-stranded DNA ends are not needed for A3G binding. The protein stoichiometry does not depend on the ssDNA substrate type, but the ssDNA length modulates the stoichiometry of A3G in the complex. We applied single molecule high-speed AFM to directly visualize the dynamics of A3G in the complexes. We were able to visualize A3G sliding and protein association-dissociation events. During sliding, A3G translocated over a 69 nucleotide ssDNA segment in less than 1 second. Association-dissociation events were more complex, as dimeric A3G could dissociate from the template as a whole, or undergo a two-step process with monomers capable of sequential dissociation. We conclude that A3G monomers, dimers, and higher order oligomers can bind ssDNA substrates independent of strand polarity and availability of free ssDNA ends.
APOBEC3G; single-stranded DNA binding proteins; site search mechanisms; atomic force microscopy; AFM; high speed-AFM
One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. However, controlled assembly of stable RNA nanoparticles with multiple functionalities which retain their original role is challenging due to refolding after fusion. Herein, we report the construction of thermodynamically stable X-shaped RNA nanoparticles to carry four therapeutic RNA motifs by self-assembly of reengineered small RNA fragments. We proved that each arm of the four helices in the X-motif can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central pRNA-X core, and each daughter RNA molecule within the nanoparticle folds into their respective authentic structures and retains their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection of ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact and strongly bound to cancers without entering the liver, lung or any other organs or tissues, while remaining in cancer tissue for more than 8 h.
Nanotechnology; Nanobiotechnology; Nanomotor; DNA packaging motor; Bacteriophage phi29; RNA nanotechnology; RNA nanoparticle; RNA therapeutics
Atomic force microscopy (AFM) is a key tool of nanotechnology with great importance in applications to DNA nanotechnology and to the recently emerging field of RNA nanotechnology. Advances in the methodology of AFM now enable reliable and reproducible imaging of DNA of various structures, topologies, and DNA and RNA nanostructures. These advances are reviewed here with emphasis on methods utilizing modification of mica to prepare the surfaces enabling reliable and reproducible imaging of DNA and RNA nanostructures. Since the AFM technology for DNA is more mature, AFM imaging of DNA is introduced in this review to provide experience and background for the improvement of AFM imaging of RNA. Examples of imaging different structures of RNA and DNA are discussed and illustrated. Special attention is given to the potential use of AFM to image the dynamics of nucleic acids at the nanometer scale. As such, we review recent advances with the use of time-lapse AFM.
Atomic force microscopy; AFM; DNA dynamics; DNA nanostructures; Holliday junctions; RNA assembly; RNA nanostructures; High-speed AFM
Exposure to organophosphorus (OP) agents can lead to learning and memory deficits. Disruption of axonal transport has been proposed as a possible explanation. Microtubules are an essential component of axonal transport. In vitro studies have demonstrated that OP agents react with tubulin and disrupt the structure of microtubules. Our goal was to determine whether in vivo exposure affects microtubule structure. One group of mice was treated daily for 14 days with a dose of chlorpyrifos that did not significantly inhibit acetylcholinesterase. Beta-tubulin from the brains of these mice was diethoxyphosphorylated on tyrosine 281 in peptide GSQQY281RALTVPELTQQMFDSK. A second group of mice was treated with a single sublethal dose of chlorpyrifos oxon (CPO). Microtubules and cosedimenting proteins from the brains of these mice were visualized by atomic force microscopy nanoimaging and by Coomassie blue staining of polyacrylamide gel electrophoresis bands. Proteins in gel slices were identified by mass spectrometry. Nanoimaging showed that microtubules from control mice were decorated with many proteins, whereas microtubules from CPO-treated mice had fewer associated proteins, a result confirmed by mass spectrometry of proteins extracted from gel slices. The dimensions of microtubules from CPO-treated mice (height 8.7 ± 3.1 nm and width 36.5 ± 15.5 nm) were about 60% of those from control mice (height 13.6 ± 3.6 nm and width 64.8 ± 15.9 nm). A third group of mice was treated with six sublethal doses of CPO over 50.15 h. Mass spectrometry identified diethoxyphosphorylated serine 338 in peptide NS338NFVEWIPNNVK of beta-tubulin. In conclusion, microtubules from mice exposed to chlorpyrifos or to CPO have covalently modified amino acids and abnormal structure, suggesting disruption of microtubule function. Covalent binding of CPO to tubulin and to tubulin-associated proteins is a potential mechanism of neurotoxicity.
chlorpyrifos; tubulin; mass spectrometry; nanoimaging; neurotoxicity
Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.
APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg2+, but dimers still predominated when Mg2+ was depleted. A His-248/His-250 Zn2+-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.
Atomic Force Microscopy; DNA-binding Protein; DNA-Protein Interaction; HIV; Protein-DNA Interaction; Single Molecule Biophysics
The dynamics of chromatin provide the access to DNA within nucleosomes and therefore, this process is critically involved into the regulation of chromatin function. However, our knowledge on the large-range dynamics of nucleosomes is limited. The questions, such as the range of opening of the nucleosome, and the mechanism whereby the opening occurs and propagates, remain unknown. Here we applied single molecule time lapse AFM imaging to directly visualize the dynamics of nucleosomes and identify the mechanism of the large range DNA exposure. With this technique, we are able to observe the process of unwrapping of nucleosomes. The unwrapping of nucleosomes proceeds from the ends of the particles, allowing for the unwrapping of DNA regions as large as dozens of base pairs. This process may lead to a complete unfolding of nucleosomes and dissociation of the histone core from the complex. The unwrapping occurs in the absence of proteins involved in the chromatin remodeling that require ATP hydrolysis for their function, suggesting that the inherent dynamics of nucleosomes can contribute to the chromatin unwrapping process. These finding shed a new light to molecular mechanisms of nucleosome dynamics and provide novel hypotheses to the understanding of the action of remodeling proteins as well as other intracellular systems in the chromatin dynamics.
To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.
This paper describes protocols for studies of structure and dynamics of DNA and protein-DNA complexes with AFM utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein-DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein-DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI-DNA complexes. When necessary, additional specifics are added to the corresponding example.
Atomic force microscopy (AFM); Force microscopy; DNA dynamics; Local DNA structures; Holliday junctions; DNA–protein interactions; Protein–protein interactions; Single-molecule techniques; Solution time lapse imaging
SfiI belongs to a family of restriction enzymes that function as tetramers binding two recognition regions for the DNA cleavage reaction. SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site specific binding. SfiI enzymatic action has been very well characterized. However, properties of the complex prior to the cleavage reaction are not clear yet. We applied AFM single molecule force spectroscopy to analyze the strength of interactions within the SfiI - DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and another one to the AFM probe. The complex was formed by the protein present in the solution. An alternative setup in which the protein was anchored to the surface allowed us to probe the stability of the complex formed with one duplex only in the approach-retraction experiments, with the duplex immobilized at the AFM tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy - DFS). The DFS data suggest that the dissociation reaction of SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was also instrumental in revealing the role of the 5 base pair spacer region within the palindromic recognition site on DNA-SfiI complex stability. The data show that, although the change of nonspecific sequence does not alter the position of activation barrier, it significantly changes values of the off rates.
synaptic complex; force spectroscopy; protein-DNA interactions; AFM; site-specific recognition
Misfolding and self assembly of proteins in nano-aggregates of different sizes and morphologies (nano-ensembles, primarily nanofilaments and nano-rings) is a complex phenomenon that can be facilitated, impeded, or prevented, by interactions with various intracellular metabolites, intracellular nanomachines controlling protein folding and interactions with other proteins. A fundamental understanding of molecular processes leading to misfolding and self-aggregation of proteins involved in various neurodegenerative diseases will provide critical information to help identify appropriate therapeutic routes to control these processes. An elevated propensity of misfolded protein conformation in solution to aggregate with the formation of various morphologies impedes the use of traditional physical chemical approaches for studies of misfolded conformations of proteins. In our recent alternative approach, the protein molecules were tethered to surfaces to prevent aggregation and AFM force spectroscopy was used to probe the interaction between protein molecules depending on their conformations. It was shown that formation of filamentous aggregates is facilitated at pH values corresponding to the maximum of rupture forces. In this paper, a novel surface chemistry was developed for anchoring of amyloid β (Aβ) peptides at their N-terminal moieties. The use of the site specific immobilization procedure allowed to measure the rupture of Aβ - Aβ contacts at single molecule level. The rupture of these contacts is accompanied by the extension of the peptide chain detected by a characteristic elasto-mechanical component of the force-distance curves. Potential applications of the nanomechanical studies to understanding the mechanisms of development of protein misfolding diseases are discussed.
Nanotechnology; amyloids; neurodegenerative diseases; protein aggregation; protein folding; intermolecular forces; AFM; Alzheimer’s disease
Misfolding and aggregation of proteins is a common thread linking a number of important human health problems. The misfolded and aggregated proteins are inducers of cellular stress and activators of immunity in neurodegenerative diseases. They might posses clear cytotoxic properties, being responsible for the dysfunction and loss of cells in the affected organs. Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular mechanism underlying these processes. Factors that lead to protein misfolding and aggregation in vitro are poorly understood, not to mention the complexities involved in the formation of protein nanoparticles with different morphologies (e.g. the nanopores) in vivo. A better understanding of the molecular mechanisms of misfolding and aggregation might facilitate development of the rational approaches to prevent pathologies mediated by protein misfolding. The conventional tools currently available to researchers can only provide an averaged picture of a living system, whereas much of the subtle or short-lived information is lost. We believe that the existing and emerging nanotools might help solving these problems by opening the entirely novel pathways for the development of early diagnostic and therapeutic approaches. This article summarizes recent advances of the nanoscience in detection and characterization of misfolded protein conformations. Based on these findings we outline our view on the nanoscience development towards identification intracellular nanomachines and/or multicomponent complexes critically involved in protein misfolding.
protein misfolding; protein aggregation; conformational disease; nanomedicine; atomic force microscopy; force spectroscopy; single molecule analyses
We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA)·poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops.
Here we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation.
The present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA)·poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.
The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane–mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.
We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.