Researchers in Munich have adapted their molecular force assay technique to a microfluidic system, allowing the strength of protein-DNA interactions to be measured in a chip format.
The measurement of intermolecular forces is a challenging and important task. Two well-known methods, atomic force microscopy and optical trapping, are capable of probing these forces1. However, these instrumentation-intensive techniques are not suitable for every application. A team from the Ludwig-Maximilians-Universität, Germany, headed by Hermann Gaub, a leader in the field of force spectroscopy, has now developed a technique, termed molecular force assay (MFA), to probe the strength of DNA hybridization and the effects of DNA-binding proteins2.
Schematic of the molecular force assay (adapted from Figure 1 of paper)
In MFA, a surface is functionalized with a carefully designed DNA construct, as seen in the left panel of the figure. This surface is brought into contact with another surface which is coated with neutravidin (center). The neutravidin binds the biotin at the end of the DNA construct, so when the two surfaces are separated, the DNA construct rips apart (dehybridization, right). The breakage can happen in one of two ways—if the hybridization of the “probe strand” is stronger than that of the “reference strand”, the reference strand will dehybridize, leaving behind a Cy3 tag. If the hybridization of the reference strand is stronger, the probe strand will dehybridize, leaving both a Cy3 tag and a Cy5 tag behind. Via the resulting fluorescence, it is possible to compare the hybridization strength of the probe strand to that of the known reference. DNA binding proteins which modify the hybridization strength of the probe strand can then be introduced.
In the original formulation of MFA, the surfaces were brought into contact using a piezoelectric device; however, in a recent paper in Lab on a Chip, the researchers have translated their assay into a microfluidic format. A microfluidic chip was used in which the lower layer contains sample wells and the upper layer contains control valves. By pressurizing button valves above each well, the top and bottom surfaces can be brought into contact and then separated. The authors demonstrated the effect of EcoRI binding on probe strand hybridization strength as a proof-of-concept, “paving the way for studies of currently unknown protein-DNA interactions, including those of transcription factors.”
Read this HOT article in Lab on a Chip today!
Protein–DNA force assay in a microfluidic format, Marcus Otten, Philip Wolf, and Hermann E. Gaub. DOI: 10.1039/C3LC50830G
1. K. C. Neuman and A. Nagy, Nature Methods, 2008, 5, 491-505.
2. P. M. D. Severin et al., Lab on a Chip, 2011, 11, 856-862.