Accurate nanoscale flexibility measurement of DNA and DNA-protein complexes by atomic force microscopy in liquid

Abstract

The elasticity of double-stranded DNA (dsDNA), as described by its persistence length, is critical for many biological processes, including genomic regulation. A persistence length value can be obtained using atomic force microscopy (AFM) imaging. However, most AFM studies have been done by depositing the sample on a surface using adhesive ligands and fitting the contour to a two-dimensional (2D) wormlike chain (WLC) model. This often results in a persistence length measurement that is different from the value determined using bulk and single molecule methods. We describe a method for obtaining accurate three-dimensional (3D) persistence length measurements for DNA and DNA-protein complexes by using a previously developed liquid AFM imaging method and then applying the 3D WLC model. To demonstrate the method, we image in both air and liquid several different dsDNA constructs and DNA-protein complexes that both increase (HIV-1 Vpr) and decrease (yeast HMO1) dsDNA persistence length. Fitting the liquid AFM-imaging contour to the 3D WLC model results in a value in agreement with measurements obtained in optical tweezers experiments. Because AFM also allows characterization of local DNA properties, the ability to correctly measure global flexibility will strongly increase the impact of measurements that use AFM imaging.

Fig. 1. Comparison of the measured persistence length for multiple DNA constructs imaged in liquid and in air. (a) Air imaging of pBR322 DNA. (b) Liquid imaging of pBR322 DNA. (c) DNA constructs pUC18 (black), pBR322 (blue) and mtDNA (red) imaged in liquid show a value of the persistence length which agrees with the three dimensional value of the DNA persistence length. Air imaging of pBR322 DNA (green) shows a value of the persistence length that is higher than the value of the three dimensional persistence length obtained with optical tweezers experiments. The inset represents DNA local bends. Each point (in green) along the DNA is separated by a distance L. The local angle θ between two segments (in yellow), which represent the tangent between two adjacent points is separated by a distance L. Each data point represents an average of measured angles for between 17 and 47 molecules, depending on the construct. Statistical uncertainties in each data point (SEM) are smaller than the data point in each case. Uncertainties in the persistence length value shown are 95% confidence interval from fits to eqn (1) or (2). (d) End-to-end distance measurements as a function of contour length. The data for the three DNA constructs are represented in solid black symbols. The green line represents the values from eqn (3) for DNA equilibrated in 2D. The red line represents the values from eqn (4) for DNA in 3D. The purple line represents the values from eqn (5) for DNA in 3D projected on a 2D surface. For all three equations, a value of 50 nm was used for the persistence length. The inset shows a diagram of a DNA molecule with the end-to-end distance R and the full DNA contour length L. Error bars are standard error for end-to-end distance measurements from 13 to 47 molecules.

Fig. 2. The yeast nucleolar HMGB protein HMO1 and the HIV-1 Viral protein R decrease and increase the persistence length of the DNA, respectively. (a) The apparent DNA persistence length in DNA-HMO1 complexes (lower-right inset) imaged in air (dark blue) is 39 ± 2 nm when fit to the 2D WLC (eqn (1)). The apparent DNA persistence length in DNA-HMO1 complexes (upper-left inset) imaged in liquid (light blue) is 21 ± 3 nm when fit to the 3D WLC (eqn (2)). (b) The apparent DNA persistence length in DNA-Vpr complexes (lower-right inset) imaged in air (dark purple) is 108 ± 2 nm when fit to the 2D WLC. The apparent DNA persistence length in DNA-Vpr complexes (upper-left inset) imaged in liquid (light purple) is 68 ± 5 nm when fit to the 3D WLC. Uncertainties are 95% confidence intervals from fits to the corresponding WLC model.

Fig. 3. Measurements of apparent DNA persistence length in the presence of HIV-1 Viral protein R (Vpr) proteins. (a) The binding of Vpr to DNA increases its apparent persistence length. DNA in the absence (black circle) and presence (red circle) of 0.75 nM Vpr is illustrated, along with a corresponding theoretical WLC curve, obtained from the average of three measurements, each fit to eqn (6). Uncertainty in the fit is SEM from the three measurements. The schematic diagrams represent DNA in solution and DNA in the presence of Vpr proteins (triangle symbols). A force F is exerted on both beads, allowing us to stretch the DNA and then to measure the mechanical properties of DNA. (b) The apparent DNA persistence length as a function of concentration is fit to the McGhee–von Hippel binding isotherm to obtain ω = 18 ± 3, n = 13 ± 4, and K_D = 0.46 ± 0.11 nM. Error bars are SEM for 3 or more measurements. Uncertainties in the fit parameters were determined from fits to eqn (7) and (8) using the χ² + 1 method.