High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins.

Vif (viral infectivity factor) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to disrupt the antiviral activity of host APOBEC3 (apolipoprotein B mRNA-editing enzyme catalytic subunit 3) proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and core-binding factor subunit ß, forming a four-protein complex called VCBC. The structure of VCBC-Cullin5 has recently been solved by X-ray crystallography, and, using molecular dynamics simulations, the dynamics of VCBC have been characterized. Here, we applied time-lapse high-speed atomic force microscopy to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of this complex, which we identified as the triangle, dumbbell, and globular structures. Moreover, we characterized the dynamics of each of these structures. Our data revealed the very dynamic behavior of all of them, with the triangle and dumbbell structures being the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and may support efforts to improve HIV treatment, because Vif is essential for virus survival in the cell.

Molecular Model for the Surface-Catalyzed Protein Self-Assembly.

The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer's and Parkinson's diseases. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting that interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind the on-surface aggregation process remain unclear. Here, we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This physical-chemical theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic α-synuclein protein and non-amyloidogenic cytosine deaminase APOBEC3G.

Insight into dynamics of APOBEC3G protein in complexes with DNA assessed by high speed AFM.

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zing-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has lacked, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built a computational model for the full-length A3G monomer and validated its structure by data obtained from time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM was applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we proposed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function.

The Enzymatic Activity of APOBE3G Multimers.

APOBEC3G (A3G) belongs to the family of cytosine deaminases that play an important role in the innate immune response. Similar to other, two-domain members of the APOBEC family, A3G is prone to concentration-dependent oligomerization, which is an integral for its function in the cell. It is shown that oligomerization of A3G is related to the packing mechanism into virus particle and, is critical for the so-called roadblock model during reverse transcription of proviral ssDNA. The role of oligomerization for deaminase activity of A3G is widely discussed in the literature; however, its relevance to deaminase activity for different oligomeric forms of A3G remains unclear. Here, using Atomic Force Microscopy, we directly visualized A3G-ssDNA complexes, determined their yield and stoichiometry and in parallel, using PCR assay, measured the deaminase activity of these complexes. Our data demonstrate a direct correlation between the total yield of A3G-ssDNA complexes and their total deaminase activity. Using these data, we calculated the relative deaminase activity for each individual oligomeric state of A3G in the complex. Our results show not only similar deaminase activity for monomer, dimer and tetramer of A3G in the complex, but indicate that larger oligomers of A3G retain their deaminase activity.

Computational Model and Dynamics of Monomeric Full-Length APOBEC3G.

APOBEC3G (A3G) is a restriction factor that provides innate immunity against HIV-1 in the absence of viral infectivity factor (Vif) protein. However, structural information about A3G, which can aid in unraveling the mechanisms that govern its interactions and define its antiviral activity, remains unknown. Here, we built a computer model of a full-length A3G using docking approaches and molecular dynamics simulations, based on the available X-ray and NMR structural data for the two protein domains. The model revealed a large-scale dynamics of the A3G monomer, as the two A3G domains can assume compact forms or extended dumbbell type forms with domains visibly separated from each other. To validate the A3G model, we performed time-lapse high-speed atomic force microscopy (HS-AFM) experiments enabling us to get images of a fully hydrated A3G and to directly visualize its dynamics. HS-AFM confirmed that A3G exists in two forms, a globular form (∼84% of the time) and a dumbbell form (∼16% of the time), and can dynamically switch from one form to the other. The obtained HS-AFM results are in line with the computer modeling, which demonstrates a similar distribution between two forms. Furthermore, our simulations capture the complete process of A3G switching from the DNA-bound state to the closed state. The revealed dynamic nature of monomeric A3G could aid in target recognition including scanning for cytosine locations along the DNA strand and in interactions with viral RNA during packaging into HIV-1 particles.

Nanoscale Characterization of Interaction of APOBEC3G with RNA.

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of the HIV-1 virus in the absence of viral infectivity factor (Vif). The molecular mechanism of A3G antiviral activity is primarily attributed to deamination of single-stranded DNA (ssDNA); however, the nondeamination mechanism also contributes to HIV-1 restriction. The interaction of A3G with ssDNA and RNA is required for its antiviral activity. Here we used atomic force microscopy to directly visualize A3G-RNA and A3G-ssDNA complexes and compare them to each other. Our results showed that A3G in A3G-RNA complexes exists primarily in monomeric-dimeric states, similar to its stoichiometry in complexes with ssDNA. New A3G-RNA complexes in which A3G binds to two RNA molecules were identified. These data suggest the existence of two separate RNA binding sites on A3G. Such complexes were not observed with ssDNA substrates. Time-lapse high-speed atomic force microscopy was applied to characterize the dynamics of the complexes. The data revealed that the two RNA binding sites have different affinities for A3G. On the basis of the obtained results, a model for the interaction of A3G with RNA is proposed.

Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy.

This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein-DNA complexes, including recent advances in the visualization of protein-DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein-DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein-DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described.

Single-Molecule Force Spectroscopy Studies of APOBEC3A-Single-Stranded DNA Complexes.

APOBEC3A (A3A) inhibits the replication of a range of viruses and transposons and might also play a role in carcinogenesis. It is a single-domain deaminase enzyme that interacts with single-stranded DNA (ssDNA) and converts cytidines to uridines within specific trinucleotide contexts. Although there is abundant information that describes the potential biological activities of A3A, the interplay between binding ssDNA and sequence-specific deaminase activity remains controversial. Using a single-molecule atomic force microscopy spectroscopy approach developed by Shlyakhtenko et al. [(2015) Sci. Rep. 5, 15648], we determine the stability of A3A in complex with different ssDNA sequences. We found that the strength of the complex is sequence-dependent, with more stable complexes formed with deaminase-specific sequences. A correlation between the deaminase activity of A3A and the complex strength was identified. The ssDNA binding properties of A3A and those for A3G are also compared and discussed.

APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies.

APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3'-5' exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.

Chromatin imaging with time-lapse atomic force microscopy.

Time-lapse atomic force microscopy (AFM) is widely used for direct visualization of the nanoscale dynamics of various biological systems. The advent of high-speed AFM instrumentation made it possible to image the dynamics of proteins and protein-DNA complexes within millisecond time range. This chapter describes protocols for studies of structure and dynamics of nucleosomes with time-lapse AFM including the high-speed AFM instrument. The necessary specifics for the preparation of chromatin samples for imaging with AFM including the protocols for the surface preparation are provided.

Interaction of APOBEC3A with DNA assessed by atomic force microscopy.

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.

Visualization of DNA and protein-DNA complexes with atomic force microscopy.

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 purification of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein-DNA complexes.

Atomic force microscopy studies of APOBEC3G oligomerization and dynamics.

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.

Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy.

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.

Ultrastable synergistic tetravalent RNA nanoparticles for targeting to cancers.

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.

Nanoscale structure and dynamics of ABOBEC3G complexes with single-stranded DNA.

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. 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 <1 s. 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 in a manner independent of strand polarity and availability of free ssDNA ends.

Specificity of binding of single-stranded DNA-binding protein to its target.

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 Mg(2+) cations or a high ionic strength. In the absence of Mg(2+) 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 Mg(2+) cations.

Imaging of nucleic acids with atomic force microscopy.

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.

Fabrication of stable and RNase-resistant RNA nanoparticles active in gearing the nanomotors for viral DNA packaging.

Both DNA and RNA can serve as powerful building blocks for bottom-up fabrication of nanostructures. A pioneering concept proposed by Ned Seeman 30 years ago has led to an explosion of knowledge in DNA nanotechnology. RNA can be manipulated with simplicity characteristic of DNA, while possessing noncanonical base-pairing, versatile function, and catalytic activity similar to proteins. However, standing in awe of the sensitivity of RNA to RNase degradation has made many scientists flinch away from RNA nanotechnology. Here we report the construction of stable RNA nanoparticles resistant to RNase digestion. The 2'-F (2'-fluoro) RNA retained its property for correct folding in dimer formation, appropriate structure in procapsid binding, and biological activity in gearing the phi29 nanomotor to package viral DNA and producing infectious viral particles. Our results demonstrate that it is practical to produce RNase-resistant, biologically active, and stable RNA for application in nanotechnology.