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.
Highlights
IntroductionSince the seminal crystallography work of Franklin and Gosling[1] revealed the double helical structure of DNA, characterization of the heterogeneous polymeric structures of DNA has been carried out using a suite of biophysical techniques including x-ray crystallography,[2,3,4] electron microscopy,[5,6,7,8,9] nuclear magnetic resonance (NMR),[10,11,12] F€orster resonance energy transfer (FRET),[13,14,15,16] and optical and magnetic tweezers.[17,18,19,20] limitations in spatial resolution without the requirement for ensemble averaging or labeling have limited the scope for high-resolution studies of the structure of DNA on flexible, individual molecules
This review provides an overview of how innovation and optimization in Atomic force microscopy (AFM) imaging have advanced our understanding of DNA structure, mechanics, and interactions
The relatively simple sample preparation, coupled with the ability to image under physiological conditions and the possibility of dynamic imaging, provides AFM with a huge degree of versatility amongst conventional microscopy techniques
Summary
Since the seminal crystallography work of Franklin and Gosling[1] revealed the double helical structure of DNA, characterization of the heterogeneous polymeric structures of DNA has been carried out using a suite of biophysical techniques including x-ray crystallography,[2,3,4] electron microscopy,[5,6,7,8,9] nuclear magnetic resonance (NMR),[10,11,12] F€orster resonance energy transfer (FRET),[13,14,15,16] and optical and magnetic tweezers.[17,18,19,20] limitations in spatial resolution without the requirement for ensemble averaging or labeling have limited the scope for high-resolution studies of the structure of DNA on flexible, individual molecules. AFM was first proposed by Binnig et al.,[21] and within 18 months, the technique was used to accomplish atomic-scale imaging of crystalline surfaces.[22] AFM is a force-based SPM technique, which reconstructs an image of the topography of a sample. This facilitates the physical analysis of samples without additional staining or labeling processes. A topographic image is generated by the scanning of a sharp nanometersized probe (grown or etched on the free end of a cantilever), which “feels” the contours of the sample surface, analogous to the tactile reading of braille.
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