1 research outputs found
Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers
Biosensors,
in which binding of ligands is detected through changes in the optical
or electrochemical properties of a DNA layer confined to the sensor
surface, are important tools for investigating DNA interactions. Here,
we investigate if conformational changes induced in surface-attached
DNA molecules upon ligand binding can be monitored by the quartz crystal
microbalance with dissipation (QCM-D) technique. DNA duplexes
containing 59–184 base pairs were formed on QCM-D crystals
by stepwise assembly of synthetic oligonucleotides of designed base
sequences. The DNA films were exposed to the cationic polyamines spermidine
and spermine, known to condense DNA molecules in bulk experiments,
or to the recombination protein Rad51, known to extend the DNA helix.
The binding and dissociation of the ligands to the DNA films were
monitored in real time by measurements of the shifts in resonance
frequency (Δ<i>f</i>) and in dissipation (Δ<i>D</i>). The QCM-D data were analyzed using a Voigt-based model
for the viscoelastic properties of polymer films in order to evaluate
how the ligands affect thickness and shear viscosity of the DNA layer.
Binding of spermine shrinks all DNA layers and increases their viscosity
in a reversible fashion, and so does spermidine, but to a smaller
extent, in agreement with its lower positive charge. SPR was used
to measure the amount of bound polyamines, and when combined with
QCM-D, the data indicate that the layer condensation leads to a small
release of water from the highly hydrated DNA films. The binding of
Rad51 increases the effective layer thickness of a 59bp film, more
than expected from the know 50% DNA helix extension. The combined
results provide guidelines for a QCM-D biosensor based on ligand-induced
structural changes in DNA films. The QCM-D approach provides high
discrimination between ligands affecting the thickness and the structural
properties of the DNA layer differently. The reversibility of the
film deformation allows comparative studies of two or more analytes
using the same DNA layer as demonstrated here by spermine and spermidine