Atomic force microscopy-A tool for structural and translational DNA research

Abstract

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated-providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.

FIG. 1.

An illustration depicting an AFM cantilever-tip probing a DNA-protein complex in fluid.

FIG. 2.

Schematic showing three key AFM imaging modes. For each mode, a cantilever-tip is raster scanned across a sample (dashed line). Surface features induce a change in the bending of the cantilever and therefore deflection of the incident laser, which is monitored by a quadrant photodiode. These changes are fed into a feedback loop to control tip-sample separation and provide a topographical map of the surface. In contact mode (i), the tip scans laterally without interrupting tip-sample contact, resulting in increased lateral forces. In dynamic modes such as tapping mode (ii) and PeakForce tapping mode (iii), the cantilever is driven to oscillate sinusoidally, resulting in intermittent contact with the surface and reduced lateral forces. In tapping mode, the cantilever is driven and oscillated close to its resonant frequency through a small amplitude of oscillation. In PeakForce tapping mode, the cantilever is driven at frequencies much lower than that of its resonant frequency through a larger amplitude of oscillation.

FIG. 3.

Timeline showing the progress of DNA imaging by AFM, from early images of DNA in air to high-resolution mapping in fluid. (a) DNA plasmids imaged in (i) air [Reprinted with permission from Bustamante et al., “Circular DNA molecules imaged in air by scanning force microscopy,” Biochemistry 31, 22–26 (1992). Copyright 1992 American Chemical Society]. (ii) aqueous solution [Reprinted with permission from Hansma et al., “Atomic force microscopy of DNA in aqueous solutions,” Nucl. Acids Res. 21(3), 505–512 (1993). Copyright 1993 Oxford University Press]. (iii) Immobilized on a cationic supported surfactant bilayer [Reprinted with permission from Mou et al., “High-resolution atomic-force microscopy of DNA: the pitch of the double helix,” FEBS Lett. 371(3), 279–282 (1995). Copyright 1995 John Wiley and Sons]. (iv) Immobilized using Ni²⁺ cations [Reprinted with permission from H. G. Hansma and D. E. Laney, “DNA binding to mica correlates with cationic radius: Assay by atomic force microscopy,” Biophys. J. 70(4), 1933–1939 (1996). Copyright 1996 Elsevier]. (v) Immobilized on APTES-functionalized mica [Reprinted with permission from Y. L. Lyubchenko, “DNA structure and dynamics: An atomic force microscopy study,” Cell Biochem. Biophys. 41, 75–98 (2004). Copyright 2004 Springer Nature]. High-resolution AFM images of the DNA helical structure, able to discern; (vi) the handedness of individual DNA molecules [Reprinted with permission from Leung et al., “Atomic force microscopy with nanoscale cantilevers resolves different structural conformations of the DNA double helix,” Nano Lett. 12, 3846–3850 (2012). Copyright 2012 American Chemical Society]; (vii) individual phosphate groups in the DNA backbone. [Reprinted with permission from Ido et al., “Beyond the helix pitch: Direct visualization of native DNA in aqueous solution,” ACS Nano, 2, 1817–1822 (2013). Copyright 2013 American Chemical Society] (viii) and kinks and defects [Reprinted with permission from Pyne et al., “Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides,” Nat. Commun. 12, 1053 (2021). Copyright 2021 Authors, licensed under a Creative Commons Attribution (CC BY) license]. (b) Schematic showing progress in AFM imaging of DNA, from low resolution imaging of molecular conformation, to double the helical structure including changes in intramolecular groove size (bracket) and defects (asterisk).

FIG. 4.

(a) Corresponding AFM images showing (i) the interaction of RNAP [Reprinted with permission from Guthold et al., “Following the assembly of RNA polymerase-DNA complexes in aqueous solutions with the scanning force microscope,” Proc. Natl. Acad. Sci. U. S. A. 91, 12927–12931 (1994). Copyright 1994 National Academy of Sciences, U.S.A.], Tp53 [Reprinted with permission from Jiao et al., “Dynamic interactions of p53 with DNA in solution by time-lapse atomic force microscopy,” J. Mol. Biol. 314(2), 233–243 (2001). Copyright 2001 Elsevier] and TOPII [Republished with permission from Alonso-Sarduy et al., “Human topoisomerase II-DNA interaction study by using atomic force microscopy,” FEBS Lett. 585(19), 3139–3145 (2011). Copyright 2011 Elsevier and Clearance Center, Inc.] with DNA and (ii) DNA architecture in the absence (-) and presence (+) of small molecule therapeutics [Reprinted with permission from Alonso-Sarduy et al., “Time-lapse AFM imaging of DNA conformational changes induced by daunorubicin,” Nano Lett. 13, 5679–5684 (2013). Copyright 2013 American Chemical Society]. [Reprinted with permission from Hou et al. “Cisplatin induces loop structures and condensation of single DNA molecules,” Nucl. Acids Res. 37, 1400–1410 (2009). Copyright 2009 Authors, licensed under a Creative Commons Attribution (CC-BY-NC) license]. [Reprinted with permission from Cassina et al., “Atomic force microscopy study of DNA conformation in the presence of drugs,” Eur. Biophys. J. 40, 59–68 (2011). Copyright 2011 Springer Nature]. (b) Schematic of DNA with bound proteins (blue) and small molecule therapeutics (purple). Protein crystal structures are shown above (PBD ID's: 5FJ8, 1TUP, and 4FM9) and small molecule therapeutics below (PDB ID's: 1DA0, 2NQ0, and 1D12).