High-speed Atomic Force Microscopy visualization of an archaeal transcription factor binding to DNA using a DNA origami frame

High-speed AFM (HS-AFM) is a unique tool that provides the high spatio-temporal resolution necessary to unravel the geometry and the mode of protein-DNA interactions, without the need for labeling. Structure and dynamics of these interactions can be directly recorded at nanometer spatial and sub-100 ms temporal resolutions in liquid. The insights obtained with HS-AFM into molecular dynamics of biologically relevant, functional processes surpass that of other approaches.
The unique protein-DNA interactions in this study were visualized using DNA origami. The basic principles of DNA origami involve a long single strand of DNA which serves as a scaffold, folded into desired shapes with the help of short oligonucleotides, referred to as staples. The experimental simplicity and the fidelity of this folding process has enabled the fast development of this area in the recent years. In this study, surface-immobilized DNA origami nanostructures are utilized to visualize individual protein molecules as they interact with dsDNA containing their binding site and attached into a DNA origami frame.
The protein of interest was a TetR-family transcriptional regulator of a large cluster of genes encoding fatty acid metabolism functions in the archaeal model organism Sulfolobus acidocaldarius (Wang et al. 2019). This regulator, FadRSa, is capable of repressing the expression of a large 23-gene cluster by interacting with only four binding sites. These binding sites are unusual in their positions, with sites at more than 130 bp upstream of the corresponding promoter. This indicates that FadRSa employs a unique repression mechanism, involving long-range interactions between its different semi-palindromic binding sites. Moreover, dimers of the protein bound to opposite sides of the DNA helix can interact via weak electrostatic interactions and form dimer-of-dimer complexes.
DNA origami frames were designed in silico and constructed, with two DNA duplexes with FadRSa binding sites incorporated into the frames. FadR was able to mediate a connection between two relaxed 74-mer duplexes inside the frame. Qualitative analysis of loose DNA duplexes with AFM height images showed X-shaped structures where two strands interact but not cross each other, only in the presence of FadRSa. These findings were further confirmed by HS-AFM visualization in liquid to mimic physiological conditions, indicating that FadRSa is able to interact with two binding sites and switch between binding as a dimer and as a dimer-of-dimers. We have demonstrated that the use of HS-AFM with the DNA origami methodology is a robust approach to study dynamic protein-DNA interactions at a single-molecule level.