The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.

Atomic force microscopy based techniques have been used in the investigation of protein folding/unfolding in order to study the protein properties at the single molecule level. These experiments are performed in buffer solution, where the AFM cantilever is used to exert and measure the mechanical forces on the protein molecules under study. Due to the motion of the cantilever relative to the liquid, a viscous drag force, which can be significant when the liquid is viscous or the cantilever's speed is high, acts on the cantilever. The irregular shape of the liquid chamber and cantilever, and the special boundary conditions of the fluid make this problem complicated and difficult to solve analytically. We measured the viscous drag forces on different cantilevers for several different cantilever speeds in solutions of different viscosities. The results show that the viscous drag on a cantilever is determined by its geometry, its relative speed, the viscosity of the solution, and the separation between the cantilever and the sample surface. This method will be useful to make corrections to the unfolding force data of proteins as well as that of AFM based force measurements.