Atomic Force Microscopy of DNA and DNA-Protein Interactions.

Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometer resolution. For biological applications, one of its key advantages is its ability to visualize the substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and its interactions with proteins.

PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy.

DNA-protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA-protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000-2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show - through the use of biotinylated DNA and streptavidin - that this selective adsorption extends to DNA-protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.

Quantification of Biomolecular Dynamics Inside Real and Synthetic Nuclear Pore Complexes Using Time-Resolved Atomic Force Microscopy.

Over the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometer length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analyzing data recorded with 50-200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an autocorrelation analysis to quantify time scales of observed dynamics and to assess their veracity-an analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

A Programmable DNA Origami Platform for Organizing Intrinsically Disordered Nucleoporins within Nanopore Confinement.

Nuclear pore complexes (NPCs) form gateways that control molecular exchange between the nucleus and the cytoplasm. They impose a diffusion barrier to macromolecules and enable the selective transport of nuclear transport receptors with bound cargo. The underlying mechanisms that establish these permeability properties remain to be fully elucidated but require unstructured nuclear pore proteins rich in Phe-Gly (FG)-repeat domains of different types, such as FxFG and GLFG. While physical modeling and in vitro approaches have provided a framework for explaining how the FG network contributes to the barrier and transport properties of the NPC, it remains unknown whether the number and/or the spatial positioning of different FG-domains along a cylindrical, ∼40 nm diameter transport channel contributes to their collective properties and function. To begin to answer these questions, we have used DNA origami to build a cylinder that mimics the dimensions of the central transport channel and can house a specified number of FG-domains at specific positions with easily tunable design parameters, such as grafting density and topology. We find the overall morphology of the FG-domain assemblies to be dependent on their chemical composition, determined by the type and density of FG-repeat, and on their architectural confinement provided by the DNA cylinder, largely consistent with here presented molecular dynamics simulations based on a coarse-grained polymer model. In addition, high-speed atomic force microscopy reveals local and reversible FG-domain condensation that transiently occludes the lumen of the DNA central channel mimics, suggestive of how the NPC might establish its permeability properties.

Nanopore extended field-effect transistor for selective single-molecule biosensing.

There has been a significant drive to deliver nanotechnological solutions to biosensing, yet there remains an unmet need in the development of biosensors that are affordable, integrated, fast, capable of multiplexed detection, and offer high selectivity for trace analyte detection in biological fluids. Herein, some of these challenges are addressed by designing a new class of nanoscale sensors dubbed nanopore extended field-effect transistor (nexFET) that combine the advantages of nanopore single-molecule sensing, field-effect transistors, and recognition chemistry. We report on a polypyrrole functionalized nexFET, with controllable gate voltage that can be used to switch on/off, and slow down single-molecule DNA transport through a nanopore. This strategy enables higher molecular throughput, enhanced signal-to-noise, and even heightened selectivity via functionalization with an embedded receptor. This is shown for selective sensing of an anti-insulin antibody in the presence of its IgG isotype.Efficient detection of single molecules is vital to many biosensing technologies, which require analytical platforms with high selectivity and sensitivity. Ren et al. combine a nanopore sensor and a field-effect transistor, whereby gate voltage mediates DNA and protein transport through the nanopore.

Determining the Effective Density and Stabilizer Layer Thickness of Sterically Stabilized Nanoparticles.

A series of model sterically stabilized diblock copolymer nanoparticles has been designed to aid the development of analytical protocols in order to determine two key parameters: the effective particle density and the steric stabilizer layer thickness. The former parameter is essential for high resolution particle size analysis based on analytical (ultra)centrifugation techniques (e.g., disk centrifuge photosedimentometry, DCP), whereas the latter parameter is of fundamental importance in determining the effectiveness of steric stabilization as a colloid stability mechanism. The diblock copolymer nanoparticles were prepared via polymerization-induced self-assembly (PISA) using RAFT aqueous emulsion polymerization: this approach affords relatively narrow particle size distributions and enables the mean particle diameter and the stabilizer layer thickness to be adjusted independently via systematic variation of the mean degree of polymerization of the hydrophobic and hydrophilic blocks, respectively. The hydrophobic core-forming block was poly(2,2,2-trifluoroethyl methacrylate) [PTFEMA], which was selected for its relatively high density. The hydrophilic stabilizer block was poly(glycerol monomethacrylate) [PGMA], which is a well-known non-ionic polymer that remains water-soluble over a wide range of temperatures. Four series of PGMA x -PTFEMA y nanoparticles were prepared (x = 28, 43, 63, and 98, y = 100-1400) and characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and small-angle X-ray scattering (SAXS). It was found that the degree of polymerization of both the PGMA stabilizer and core-forming PTFEMA had a strong influence on the mean particle diameter, which ranged from 20 to 250 nm. Furthermore, SAXS was used to determine radii of gyration of 1.46 to 2.69 nm for the solvated PGMA stabilizer blocks. Thus, the mean effective density of these sterically stabilized particles was calculated and determined to lie between 1.19 g cm-3 for the smaller particles and 1.41 g cm-3 for the larger particles; these values are significantly lower than the solid-state density of PTFEMA (1.47 g cm-3). Since analytical centrifugation requires the density difference between the particles and the aqueous phase, determining the effective particle density is clearly vital for obtaining reliable particle size distributions. Furthermore, selected DCP data were recalculated by taking into account the inherent density distribution superimposed on the particle size distribution. Consequently, the true particle size distributions were found to be somewhat narrower than those calculated using an erroneous single density value, with smaller particles being particularly sensitive to this artifact.