Multiplexed force extension and manipulation of DNA-protein interactions
Here, multiple protein-coated DNA molecules are tethered between a bead and a glass surface. Using the AFS we can stretch the DNA molecules by pulling the beads away from the surface while measuring the z-position of each individual bead. This makes it possible to obtain the force-distance curve of many protein-coated DNA molecules in parallel.
Figure 1 shows the force-extension curve of a DNA molecule measured before (left) and after (right) the incubation of 1 μM of RecA – a protein involved in DNA repair. From the figure we can observe that RecA substantially lengthens the DNA as it forms filaments around the DNA structure, preventing it from coiling.
Figure 2 shows the normalized length-time traces of two individual DNA molecules in the presence of 0.5 μM RecA. At a constant force of 40 pN, the DNA length increases to >1.4x the contour length (Lc) because of RecA binding to the DNA. When the force is set to 2.5 pN again, the length of the DNA decreases due to RecA disassembly. This indicates that the RecA binding is strongly dependent on tension and is therefore enhanced by increased force. From the figure, we can also observe a slightly different behavior between the two molecules which underlies the importance of obtaining many single-molecule measurements.
Highly parallel measurements of DNA-protein interactions typically require that both constant and dynamic forces can be applied on the DNA. A high force and distance resolution and the ability to apply hundreds of picoNewtons to the DNA molecule are necessary to obtain the complete force-distance curve.