Measuring the binding affinity of Ku70/80 to dsDNA in a microfluidic system using the Fluidity One-W
Collaboration with Paloma F. Varela from JB Charbonnier team at I2BC/ Cea Saclay, Gif-sur-Yvette, France.
V. Denninger, H. Chouhery & Sebastian Fiedler at Fluidic Analytics.
If left unresolved, DNA double strand breaks (DSB) can lead to severe genomic mutations that can result in cell death or cancer. The heterodimer, Ku70/80, is an early player in DSB repair and is part of the non-homologous end joining (NHEJ) repair pathway. Ku70/80 binds to both ends of the dsDNA at the damaged sites, acting as a marker to recruit other repair proteins required for NHEJ.
Figure 1: Equilibrium binding curve of Ku70/80 and FAM-labeled dsDNA oligonucleotide (18 bp). Measurements were performed in triplicate. KD was determined by non-linear least squares fitting.
Using the Fluidity One-W, we measured the KD value of Ku70/80 binding to DNA, fully in solution, by diffusional sizing of the protein–DNA complex in a microfluidic system. Increasing concentrations of Ku70/80 were titrated against 10 nM of an 18 bp dsDNA oligonucleotide which was labeled at the 5’ end with FAM (fluorescein amidite). The resulting KD value of ~2 nM revealed a high affinity of the Ku70/80 heterodimer to dsDNA, reflecting its crucial role in DSB repair (Figure 1).
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