DNase Cleavage of Lambda DNA
DNase1 is an enzyme that nonspecifically cleaves DNA. Here, the Cypher VRS was used to monitor the action of DNase1 on lambda DNA. Briefly, the DNA was bound to a mica substrate with MgCl2 buffer, and DNase was introduced. Images were obtained in tapping mode at a line rate of 625 Hz with 320×64 pixels using an Olympus AC10 probe for a frame rate of 8.7 frames per second (fps). Beside the movie, we’ve selected three key frames taken over an interval of just 2.5 seconds. In the first image (top), several DNA strands are observed. In the second image (center), note the DNase enzyme bound to one of the overlapping strands (circle). In the third and last image (bottom), this strand has been cleaved at the binding point.
Assembly of Collagen Fibrils on Mica
Collagen is the most abundant protein found in humans and other animals. It assembles in vivo into hierarchical structures found in numerous tissues. In addition, it is widely used to create substrates and 3D scaffolds in vitro for cell and tissue culture. Hence, understanding the mechanism of collagen self-assembly has both fundamental and practical importance.
The results shown here were obtained on a Cypher VRS video-rate AFM. Briefly, the movie begins with images of bare mica in buffer obtained in tapping mode at a line rate of 400 Hz with 512×256 pixels for a frame rate of 1.5 fps. A dilute solution of collagen molecules is added. The three steps of assembly are captured: (1) Adsorption of highly mobile collagen molecules on mica within several seconds after injection; (2) Nucleation of poorly ordered structures through the spatial fluctuations, interaction and aggregation of adsorbed collagen molecules; and (3) Growth of fibrils exhibiting the D-band structure by attachment of collagen monomers as well as merging of small nucleated collagen aggregates.
Samples provided courtesy of Jinhui Tao and Jim De Yoreo, Pacific Northwest National Laboratory.