Freshly cleaved mica is used as the substrate to deposit dsDNA and protein–DNA complexes. Because freshly cleaved mica is negatively charged (20 (link),21 (link)), Mg2+, a divalent cation, was used in the deposition buffer to promote the adhesion of negatively charged DNA to the mica surface and prevent (18 (link)) binding of HMGB proteins (21 (link),22 ). Isolated plasmid pBR322 (Fermentas) was linearized by digestion with PvuII (Fermentas) followed by phenol extraction. The DNA was diluted with 10 mM Tris–HCl (pH 8.0), 5 mM MgCl2 to 0.11 nM to avoid aggregation (23 (link)). Sample deposition involved the following steps: (i) muscovite mica (Ted Pella Inc.) was cleaved and washed with buffer containing 10 mM Tris–HCl pH 8.0, 5 mM MgCl2. (ii) The sample was air dried, rinsed with 5 ml distilled water and air dried again. (iii) To collect images of bare DNA, a volume of 7 μl of the DNA solution (0.11 nM) was deposited on the mica surface for 10 min. (iv) The surface was rinsed with 5 ml distilled water and then air dried for 10–15 min, after which excess water was removed by careful blotting. Attempts at drying with an air stream produced elongated, non-equilibrated DNA, likely due to hydrodynamic forces. Samples were then imaged within 48 h.
To collect images of protein bound to DNA, HMGB2 (Box A) and HMGB1 (Box A + B) proteins were purified as described elsewhere (1 (link)) and were incubated with dsDNA before deposition. Optimal samples for imaging and analysis required moderate concentrations of both protein and dsDNA. A volume of 1.3 μl protein solution [20 nM HMGB2 (Box A) or HMGB1 (Box A + B)] and 7 μl of 0.11 nM DNA were combined.. Thus the sample consists of 0.09 nM DNA and 3.1 nM of either HMGB2 (Box A) or HMGB1 (Box A + B). The binding sizes for HMGB2 (Box A) and HMGB1 (Box A + B) proteins may be estimated to be 7 bp and ∼18 bp, from previous studies (8 (link),9 (link),24 (link)). Therefore, the ratio of binding sites on the dsDNA to protein molecules is 18:1 and 7:1 respectively (nearly 1 protein for every 120 bp). This low protein/DNA concentration ratio allows enough dsDNA-binding sites while minimizing protein self-aggregation.
A Pico-Plus scanning probe microscope (SPM; Agilent Technology) was employed. The SPM was operated in tapping or intermittent contact mode in air. Tapping mode has been used widely for imaging soft biological samples. In this mode, the cantilever is driven at a fixed frequency (near its resonance frequency) as it scans the sample. The tip is allowed to make transient contact with the sample surface at the bottom of the oscillation, which reduces its oscillation amplitude. The amplitude is used as a height feedback control parameter. The height, controlled by a piezo-crystal voltage, is extracted during scanning to form topography images. In addition, the phase of the oscillations is also used to form images. The resolution in tapping mode can be nearly as high as in contact mode, which is much more damaging to soft samples. Background cantilever thermal noise is inversely proportional to the resonance frequency (25 ), and a cantilever with higher resonance frequency allows for a faster scanning rate. Polymer surfaces become stiffer at higher frequencies, further reducing sample damage when using a high resonant frequency cantilever. Thus, cantilevers with the highest resonant frequency are preferable. Budget Sensors 300Al reflex silicon AFM tips were employed (resonance frequency ∼300 kHz; spring constant ∼40 N/m). Tips typically have ∼10 nm radius of curvature, limiting lateral imaging resolution to ∼15 nm under the best conditions. The scan range was either 2 μm × 2 μm or 1 μm × 1 μm at 512 × 512 pixels. The scan rate was typically 2 Hz. Both topography and phase images were analyzed. DNA contours were traced semi-automatically using ImageJ software (26 ) from NIH with the NeuronJ plug-in (27 (link)). The tracing step size varied from 1 to 10 pixels. The DNA bend angle was measured at the protein-binding site using National Instruments Vision Assistant 7.0 software.
To collect images of protein bound to DNA, HMGB2 (Box A) and HMGB1 (Box A + B) proteins were purified as described elsewhere (1 (link)) and were incubated with dsDNA before deposition. Optimal samples for imaging and analysis required moderate concentrations of both protein and dsDNA. A volume of 1.3 μl protein solution [20 nM HMGB2 (Box A) or HMGB1 (Box A + B)] and 7 μl of 0.11 nM DNA were combined.. Thus the sample consists of 0.09 nM DNA and 3.1 nM of either HMGB2 (Box A) or HMGB1 (Box A + B). The binding sizes for HMGB2 (Box A) and HMGB1 (Box A + B) proteins may be estimated to be 7 bp and ∼18 bp, from previous studies (8 (link),9 (link),24 (link)). Therefore, the ratio of binding sites on the dsDNA to protein molecules is 18:1 and 7:1 respectively (nearly 1 protein for every 120 bp). This low protein/DNA concentration ratio allows enough dsDNA-binding sites while minimizing protein self-aggregation.
A Pico-Plus scanning probe microscope (SPM; Agilent Technology) was employed. The SPM was operated in tapping or intermittent contact mode in air. Tapping mode has been used widely for imaging soft biological samples. In this mode, the cantilever is driven at a fixed frequency (near its resonance frequency) as it scans the sample. The tip is allowed to make transient contact with the sample surface at the bottom of the oscillation, which reduces its oscillation amplitude. The amplitude is used as a height feedback control parameter. The height, controlled by a piezo-crystal voltage, is extracted during scanning to form topography images. In addition, the phase of the oscillations is also used to form images. The resolution in tapping mode can be nearly as high as in contact mode, which is much more damaging to soft samples. Background cantilever thermal noise is inversely proportional to the resonance frequency (25 ), and a cantilever with higher resonance frequency allows for a faster scanning rate. Polymer surfaces become stiffer at higher frequencies, further reducing sample damage when using a high resonant frequency cantilever. Thus, cantilevers with the highest resonant frequency are preferable. Budget Sensors 300Al reflex silicon AFM tips were employed (resonance frequency ∼300 kHz; spring constant ∼40 N/m). Tips typically have ∼10 nm radius of curvature, limiting lateral imaging resolution to ∼15 nm under the best conditions. The scan range was either 2 μm × 2 μm or 1 μm × 1 μm at 512 × 512 pixels. The scan rate was typically 2 Hz. Both topography and phase images were analyzed. DNA contours were traced semi-automatically using ImageJ software (26 ) from NIH with the NeuronJ plug-in (27 (link)). The tracing step size varied from 1 to 10 pixels. The DNA bend angle was measured at the protein-binding site using National Instruments Vision Assistant 7.0 software.
