Measure DNA Repair Mechanisms with High Throughput

Part of: DNA Repair

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.

AFS DNA-Protein Interactions General

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.

AFS AFS Force extension of RecA-DNA

1 Force-distance curves of DNA in the absence (left) and presence (right) of RecA.

AFS Time extension of RecA-DNA AFS

Normalized length-time traces of two individual DNA molecules in the presence of RecA. The figure shows a force-dependent binding behavior of the length increasing protein.

Data courtesy of Prof. Erwin Peterman and Prof. Gijs Wuite at the VU University Amsterdam.

Hill et al. (2017)
Bioch.Soc.Tran.

Want to learn more about the high throughput capabilities of AFS™?
Visit the DNA/RNA Structural Mechanics Page

Solutions

AFS™ Parallel Single-Molecule Force Spectroscopy
AFS

Parallel Single-Molecule Force Spectroscopy

Acoustic Force Spectrosocpy (AFS™) is a new single-molecule and single-cell manipulation method capable of applying acoustic forces on hundreds of biomolecules in parallel for precise experimentation with high throughput. It enables scientists to probe thousands of individual molecules in parallel (such as RNA, DNA, proteins, and living cells), allowing statistical analysis of the mechanical properties of biological properties of biological systems based on a single experiment.

Want to learn more?

Would you like to receive exclusive news on the latest products, single-molecule events and breakthrough science from us?


You can unsubscribe at any time from our marketing emails. By submitting the form you agree to LUMICKS' privacy policy.

Take your research to the next level.

Get exclusive news on the latest products, single-molecule events and breakthrough science.

Newsletter pop up
By clicking the subscribe button you agree to LUMICKS’ privacy policy.