Oct 29, 2020
5 Methods for DNA-protein imaging by AFM in fluid
- Philip J. Haynes1,2,3,
- Kavit H. S. Main1,4,
- Alice L Pyne5
- 1London Centre for Nanotechnology, University College London, London WC1H 0AH, UK;
- 2Molecular Science Research Hub, Department of Chemistry, Imperial College London, W12 0BZ, UK;
- 3Department of Physics and Astronomy, University College London, London, WC1E 6BT, UK;
- 4UCL Cancer Institute, University College London, London, WC1E 6DD, UK;
- 5Department of Materials Science, Sir Robert Hadfield Building, University of Sheffield, S1 3JD
Protocol Citation: Philip J. Haynes, Kavit H. S. Main, Alice L Pyne 2020. 5 Methods for DNA-protein imaging by AFM in fluid. protocols.io https://dx.doi.org/10.17504/protocols.io.bncqmavw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2020
Last Modified: October 29, 2020
Protocol Integer ID: 43120
Keywords: Atomic force microscopy, AFM, DNA, Supercoiling, Double helix, DNA-protein binding,
Abstract
This is part 5 of the "Atomic Force Microscopy of DNA and DNA-Protein Interactions" collection of protocols.
Collection Abstract: Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize 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 it’s interactions with proteins.
Guidelines
One of the major drawbacks in investigating DNA-protein interactions by AFM is that, at physiologically relevant protein concentrations, protein tends to bind nonspecifically to the underlying substrate. This ultimately obscurs the adsorbed DNAbound protein complexes. This is often the case when adopting adsorption methods that utilise divalent cations and poly-L-lysine (Fig. 5bi-ii). To minimise non-specific protein binding to the substrate, a PEGylated surface can be adopted which passivates the surface against protein adsorption.
Fig. 5 Three methods used to adsorb DNA onto a mica substrate, divalent cations, poly-L-lysine, and PLL-b-PEG. (a) Illustrations of the three methods showing DNA (grey), protein (green), positive adsorption methods (red) and passivating PEG chains (grey). In PLL-b-PEG block copolymers (aiii), the densely packed brush-like PEG chains repel proteins from the underlying substrate. (b, c) AFM topographic images of 496 base-pair linear DNA containing no protein (b) or with 200 nM PARP1 (a nuclear enzyme, seen as white blobs) (c), adsorbed by (i) divalent cations, (ii) poly-L-lysine (PLL1000–2000) and (iii) PLL-b-PEG diblock copolymers supplemented with PLL1000–2000. Adapted from ref. 31, with permission. Scale bars: 100 nm. Color scale (scale bar inset in biii): 4 nm.
On a PEGylated surface, the brush-like PEG113 chains protrude with a vertical end-to-end distance comparable to the height of a DNA molecule (see Fig. 5aiii). These brushes may interact with the tip and hinder access to the surface. When imaging on such a surface, key parameters need to be adjusted when compared to the imaging conditions described in protocol 4.
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Methods for DNA-protein imaging by AFM in fluid
Methods for DNA-protein imaging by AFM in fluid
PEGylate the substrate and adsorb DNA/DNA-protein as described in protocol 2, method 2.3. Place the sample in the AFM.
Optimise imaging parameters (protocol 4) such that the DNA and DNA-protein complexes are sufficiently tracked.
If inadequate DNA tracking persists due to the influence of the brush-like PEG chains, increase the PeakForce Setpoint (see Note 27) and PeakForce Amplitude. Adjust and optimise the Lift Height accordingly (see Note 26).
All other imaging parameters are synonymous to those stated in protocol 3 and protocol 4 when approaching and optimising. A non-exhaustive list of parameters when using an Fast-Scan D are outlined in Table 1.
| PFT Parameters | Typical value | |
| Scan Size [nm] | 120 - 250 | |
| Pixel density [pixels/line] | 256 - 512 | |
| Line rate [Hz] | 3-5 | |
| Imaging PeakForce Setpoint [pN] | ~ 70 * | |
| PF acquisition Frequency, see Note 19 [kHz] | 8 | |
| LP Deflection Bandwidth [kHz] | 20 | |
| Sync Distance, see Notes 19 and 21 [μs] | 70 / 20 | |
| PeakForce Amplitude, Note 22 [nm] | 5-10 * | |
| Lift Height, see Note 26 [nm] | 5-7 | |
| Z Range (Z Limit if using MultiMode® 8) [nm] | 500-1000 | |
| Deflection Limit [V] | ⋜12.24 |
Table 1: Typical parameters used for an FastScan-D cantilever on a FastScan Bio™ AFM system, operating in PeakForce Tapping mode
*Parameters may need to be adjusted when imaging on a surface passivated with PLL-b-PEG (protocol 2, method 2.3). The PeakForce Setpoint and PeakForce Amplitude may need to be increased to 130 pN and 20 nm respectively (protocol 5).