TY  - THES
AB  - A compact, single beam optical tweezers system for force measurements and manipulation of individual double-stranded DNA (dsDNA) molecules was integrated into a commercial inverted optical microscope. A maximal force of 350 pN combined with a force sensitivity of less than 0.5 pN allows measurements of elastic properties of single molecules which complements and overlaps the force regime accessible with atomic force microscopy (AFM). The manipulation and measurement performance of this system was tested with individual Lambda DNA molecules and renders new aspects of dynamic forces phenomena with higher precision in contrast to AFM studies. An integrated liquid handling system with a fluid cell allows investigation of the force response of individual DNA molecules in the presence of DNA binding agents.
Mechanical properties of double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical tweezers experiments with sub-piconewton force resolution. Different binding modes could unambiguously be distinguished by analyzing the mechanic response of a dsDNA molecule to an applied external force. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalator ethidium bromide, YO-1 and daunomycin as well as the bisintercalator YOYO-1 on Lambda DNA. Binding of molecules to the minor and major groove to dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the B-S transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities in the range between 100 nm/s and 12,000 nm/s were found for daunomycin YOYO-1. These time dependent mechanical properties directly reflect the kinetics of the binding and unbinding. Force dependent time constants of 0.29 - 1.34 s and 0.15 - 0.60 s were determined for YOYO-DNA complexes and daunomycin-DNA complexes, respectively.
Also we used the optical tweezer setup as an optiomechanical force transducer to analyze the rupture forces between mouse anti-chicken immunoglobulin class M (Anti-IgM) coupled to the surface of a trapped bead and membrane-bound B-cell receptors (immunoglobulin class M) on living chicken DT40 B-cells. The experiment exhibits specific single and multiple bond interactions and dissociations when an external force was applied. At a loading rate of 45 pN/s a single bond rupture force of 12.5 pN and integer multiples of this value during multiple bond rupturing could be identified. These experiments will allow interesting and important in-vitro testing of living cells in the future.
DA  - 2005
KW  - Optische Pinzette , DNS , Immunkomplex , Kraftspektroskopie , Lambda-DNA , Schmelzen der DNA , Force spectroscopy , Lambda DNA , DNA overstretching and melting
LA  - ger
PY  - 2005
TI  - Kraftspektroskopie und Einzelmoleküldetektion mit der Optischen Pinzette
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-8682
Y2  - 2024-11-22T04:53:23
ER  -