DNA-basierte molekulare Maschinen und Aktuatoren

In the emerging field of nanobiotechnology, deoxyribonucleic acid
(DNA) has already established as a key building block with
versatile properties. In the present thesis, self-organising and
controllable nanoactuators and a functional "nanomachine",
consisting of artificially synthesised short DNA strands, have been
designed and characterised. The techniques employed for the
construction and analysis of the DNA nanodevices are from the field
of physics and biochemistry. The essential results of this work
are: • Characterisation of the DNA tweezers in their open and
closed configuration by the means of single-molecule fluorescence
measurements. Hence the distribution of the transfer efficiency
could be determined for both states and the influence of salt
concentration on the distribution in the open state. Based on these
results, the distributions of the distances between the dye
molecules (and therefore the distances between both arms of the
tweezers) could be calculated. For the closed state three
subpopulations have been found. • The successful integration of
gold nanoparticles into switchable DNA actuators. Here, the
fluorescence emission of dyes was measured whose distance to a gold
nanoparticle was varied by the DNA actuator. The bulk experiments
showed that gold nanoparticles can be effectually used as quenchers
for the fluorescent dyes in DNA nanoactuators to monitor distance
changes ≥ 10 nm. • The existing concepts for the operation of
nanomechanical DNA machines could be employed for the construction
of the first switchable aptamer. Switchable aptamers fulfil the
function of binding and releasing a protein or other molecules
reversibly in a controlled way. In the present project, a molecular
machine based on a DNA aptamer was constructed that can cyclically
bind to the human protein thrombin and release it upon addition of
fuel strands. In fact, one can imagine the developed aptamer
machine as a specific "molecular claw". The mode of operation of
this switchable aptamer was verified and characterised by
fluorescence resonance energy transfer and fluorescence anisotropy
measurements. To study the kinetics of the release of thrombin in
detail the binding constant of the aptamer machine has been
determined using fluorescence correlation spectroscopy
measurements. A model of the single reaction steps has been put up,
simulated numerically and fitted to the experimentally obtained
curves. The combination of the operation principle of DNA based
nanomechanical devices with the binding properties of DNA aptamers
greatly expands the scope for the design and construction of
further functional DNA nanodevices. It is assumed that in the near
future single-molecule measurements will be increasingly employed
within the development of DNA nanomachines and actuators to gain
insight in the actually existing structures of these devices.