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Measuring the binding affinity of Ku70/80 to dsDNA using microfluidic diffusional sizing

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.

 

Background

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.

 

Ku70/Ku80 image

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.

 

Results

Using the Fluidity One-W, we measured the KD value of Ku70/80 binding to DNA, fully in solution, by microfluidic 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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