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WIREs Nanomed Nanobiotechnol
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Unraveling DNA dynamics using atomic force microscopy

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Abstract The elucidation of structure–function relationships of biological samples has become important issue in post‐genomic researches. In order to unveil the molecular mechanisms controlling gene regulations, it is essential to understand the interplay between fundamental DNA properties and the dynamics of the entire molecule. The wide range of applicability of atomic force microscopy (AFM) has allowed us to extract physicochemical properties of DNA and DNA–protein complexes, as well as to determine their topographical information. Here, we review how AFM techniques have been utilized to study DNA and DNA–protein complexes and what types of analyses have accelerated the understanding of the DNA dynamics. We begin by illustrating the application of AFM to investigate the fundamental feature of DNA molecules; topological transition of DNA, length dependent properties of DNA molecules, flexibility of double‐stranded DNA, and capability of the formation of non‐Watson–Crick base pairing. These properties of DNA are critical for the DNA folding and enzymatic reactions. The technical advancement in the time‐resolution of AFM and sample preparation methods enabled visual analysis of DNA–protein interactions at sub‐second time region. DNA tension‐dependent enzymatic reaction and DNA looping dynamics by restriction enzymes were examined at a nanoscale in physiological environments. Contribution of physical properties of DNA to dynamics of nucleosomes and transition of the higher‐order structure of reconstituted chromatin are also reviewed. WIREs Nanomed Nanobiotechnol 2011 3 574–588 DOI: 10.1002/wnan.150 This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Histone core slips along DNA. (a) Snapshots of the complex obtained using a Brownian‐dynamics simulation. (b) AFM image of reconstituted nucleosome from a 437 bp DNA chain. Scale bar: 50 nm. (c) Probability distribution of the position of histone on the DNA chain. Rp is measured from the AFM image as shown schematically in the figure.

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Dynamic enzyme–DNA interactions. Successive time‐lapse images of EcoRII–DNA complex (a) and EcoP15I–DNA complex (b) obtained at 1–2 fps. The elapsed time is shown in each image. (a) EcoRII translocation on looped DNA. Individual frames are shown every 2.5 s. As the long arm gradually becomes shorter, the loop length increases. Scale bar: 50 nm. (b) Translocation and formation of an extruded loop on an EcoP15I–DNA complex between 1 and 10 s, before release of the loop at 11 s. Scale bar: 100 nm.

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AFM analyses of various G‐quadruplex structures. (A) AFM images of linearized plasmid carrying telomeric DNA. The ‘rigid’ (open triangle), ‘branched’ (filled triangle), ‘globular’ (asterisk) structures are indicated in AFM images and summarized in the table together with the ‘stretched’ form. The frequencies of each form in various types of DNA are shown below in the absence or presence of 100 mm K+. Scale bars: 100 nm. (B) Zoomed AFM images of G‐quadruplex structures with corresponding diagrammatic representations of the DNA arrangement. Areas (140 × 140 nm2) showing regions of transcribed plasmids containing loops (a, b), a blob (c), and a spur (d). The blue lines in the diagrams represent the noncoding strand of the DNA, the red lines represent the G‐rich coding DNA strand, and the green lines represent hybridized mRNA (Reproduced with permission form Ref 57. Copyright 2009 Oxford University Press).

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Analysis of the movement of plasmid DNA under fluid. (a) Successive time‐lapse images of a pGEMEX‐1 obtained at 2 fps. The elapsed time is shown in each image. Scale bar: 200 nm. (b) DNA strands from the 10 successive images were overlaid to show DNA movement. Scale bar: 200 nm. The fast‐scanning (parallel to x‐axis) and slow scanning directions (parallel to y‐axis) are shown by the blue and red arrows, respectively.

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Microscopic observations of a single T4 DNA chain. (a) FM image. (b) AFM image of the very same DNA as in (a). (c) Coarse‐grained image of (b) using a Gaussian filter of ∼300 nm. (d) Log–log plot of the end‐to‐end distance versus contour length along the trail of the DNA chain in (b). The dotted lines have slopes of 1.0, 0.5, and 0.75 (left to right), respectively. (e) Transmission electron microscopic image of spermidine‐collapsed T4 DNA.

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Relationship between the twisting number and the writhing number at a constant linking number. The linking number (Lk) is expressed as a sum of the twisting number (T) and the writhing number (W). Without any strand break or nicking, the linking number remains constant (dotted line), and thus, the superhelical strain is distributed between twisting and writhing. The local binding of RepE54 dramatically shifts the distribution from writhing to twisting and promotes a plasmid relaxation (arrow).

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Applicability of AFM in biology. The wide range of AFM applicability is suitable for structural analyses of various types of DNA and DNA–protein complexes.

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Theoretical free energy F of a DNA molecule interacting with a core protein. (a) Nonlinear coupling between bending and twisting is taken into account on the simulation of the nucleosome model. C is as an index of wrapping degree. It is noted that proper left‐handed wrapping is caused only for the core with medium sized core. The solid, dotted, and broken curves correspond to core sizes σc = 1 : 4, σc = 1 : 5, and σc = 1 : 6, respectively. (b) AFM image of reconstituted chromatin. The 106 kbp supercoiled plasmid DNA was used as a template DNA. Scale bar: 250 nm.

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Schematic illustration of the principles and mode of AFM. (a) The principles of AFM operation. A sharp probe mounted at the end of a flexible cantilever raster‐scans over the sample surface in a series of horizontal sweeps.The surface topology was then reconstructed from the vertical movement of the sample stage (piezoscanner) which was controlled by the feed‐back system. The vertical displacement of the probe during scanning is monitored using the optical lever method, in which the small displacement (down to 0.1 nm) of the cantilever can be amplified and detected by the position sensitive photo‐detector. (b)–(d) Schematic illustrations of the imaging modes of AFM together with the representative images of DNA molecules. (b) In contact mode, the cantilever is dragged across the sample surface. AFM image shows 10 kbp double‐stranded circular plasmid DNA. Scale bar: 200 nm. (c) In Tapping Mode™, the cantilever is oscillated at its resonance frequency. AFM image shows 14 kbp double‐stranded linear DNA. Scale bar: 200 nm. (d) In noncontact mode (frequency modulation mode), the tip of the cantilever does not contact the sample surface. The cantilever is oscillated at a frequency slightly above its resonant frequency and the van der Waals forces or any other long range force which extends above the surface is detected by the decrease of the resonance frequency of the cantilever. AFM image shows a DNA tile with scan size of 38 × 38 nm2 (Reproduced with permission from Ref 31. Copyright 2010 Japan Society of Applied Physics).

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Phase transition in reconstituted chromatin. Quasi‐three‐dimensional FM images (a)–(c), corresponding two‐dimensional images (insets) and AFM images (d)–(f) of reconstituted chromatin adsorbed onto a mica surface. The images in Figure 1 (a, d), (b, e), and (c, f) are of exactly the same reconstituted chromatin. The samples of reconstituted chromatin were prepared from core histones and 106 kbp plasmids (circular DNA). The mass ratio [histone]/[DNA] is 1.0 in (a, d) and 1.3 in (b, c, e, and f). In (d), nucleosomes are dispersed in the reconstituted chromatin, whereas in (e) the condensed and dispersed parts coexist. In (f) the reconstituted chromatin is entirely condensed.

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