Current Research Highlights
Optical Tweezers: At the Institute of Physics, a new type of combined optical tweezers instrument
has been setup in our group (2, 3, 7). The implementation of confocal optical spectroscopy with single
photon sensitivity allows combining force measurement in the subpiconewton regime with optical detection
of nanometer distances between single fluorophores (e.g. fluorescence resonance energy transfer, FRET) on
interacting biomolecules or detection of local motion on a biomolecule. In figure 1 part of the current
setup is shown.
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Figure 1: The biomolecule of interest is placed inbetween two polystyrene spheres (size ~2 µm
diameter). The lower bead is held by a micropipette by suction and the top bead is held by laser
tweezers that allow measuring sub-pN forces. On one end, the molecule is covalenty attached and
on the other end, the molecule is bound via a strong biomolecular bond. Optical spectroscopy is
performed by illuminating part of the field of view by an excitation laser (488 nm) and the optical
response (fluorescence) is read out by EM-CCD or APDs. |
By fluorescently labeling molecules (e.g. dsDNA with SYBR® green, or single fluorophores at
specific sites) the mechanical response can be evaluated with pN precision and in parallel visual inspection
can take place by single molecule fluorescence imaging (see figure 2). Our current measurements suggest that
dsDNA undergoes a structural transition since the fluorescence response does not change during the transition
at pulling forces between 65 and 75pN.
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Figure 2: A single dsDNA molecule subjected to an external force of ~80 pN. The dsDNA
molecule is clamped in between two polystyrene spheres and pulled by a piezo electric element. One
question remaining is: Does the dsDNA dehybridize (dsDNA to two ssDNA) during the overstretch transition
or does it undergo a structural transition into S-form (as shown in the inset: data from molecular
modeling Lebrun A et al. NAR (1996)).
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Additional projects are focused on the genetic transfer from bacteria to plant cells and at influences on
DNA structure/mechanics of substances used for gene transfer in eukaryotes. Here we study binding kinetics
or mechanical changes induced by binding the molecules of interest to DNA molecules and then correlate our
physical measurement to structure and function of the molecules involved in the process (collaborations with
the
Engel group and
Giese group.).
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