Although the successful force-extension measurement on a DNA carpet shown in Fig.4.15 was a single event, the fact that the force drops back to zero at the contour length of λ-DNA supports strongly the idea that the force was generated by DNA.
This observation confirms the feasibility of stretching a carpet of DNA with a simple force apparatus. For combined measurements of force and birefringence still a lot of problems have to be solved. The first point is to clarify the reason of why the DNA breaks. The fact that the breakage is reversible indicates that the tether itself breaks.
Investigation with fluorescence microscopy showed that the streptavidin/biotin link-age broke [51]. To see this, a spherically shaped glass surface14 coated with strepta-vidin was brought in contact with a gold covered coverslip on which DNA has been grafted by the thiol/gold chemistry; the free end had been functionalized with biotin.
The thickness of the gold layer was less than 20 nm enabling an observation of the YOYO-1 labelled DNA. Because of the spherical shape there was a region were the distance between gold surface and glass is in the order of the thickness of the DNA carpet of about 1µm and thus one expects to obtain double-end-grafted DNA. In-deed by moving the upper glass with a motor a couple of molecules were stretched.
When the DNA was fully stretched, the tether at the streptavidin-coated glass sur-face broke. Surprisingly there were molecules which were able to bind again to the streptavidin surface even though the tether has been broken before. This could ex-plain the observation made with the force apparatus why we observed a breakage of the molecules and why the force could be measured again. To enhance the stability of the streptavidin/biotin linkage one could functionalize each DNA molecules with multiple biotins with a technique described in [17].
The second point for a successful investigation of the B-S transition is the force apparatus itself. Because of the bad reproducibility of the experiment a lot of im-provements are required for routine force measurements. One important point is the possibility of grafting the DNA inside the sample cell in order to preserve the DNA from being damaged by shear flows. Furthermore the stability of the system has to be improved. A more compact design would be desirable what can be achieved with smaller cantilever. For a cantilever of a length of 7 mm and a width of 1 mm the thick-ness has to be 30µm to get a spring constant of 1 N/m. Such thin glass is provided by Schott and it has been shown that one can achieve a force resolution of ∼10nN
14The spherical shape were created by melting a glass rod.
with such thin cantilever and fiber-interferometric force measurements [155].
.
The goal of the present work was to develop chemical, molecular biological and phys-ical methods for stretching an ensemble of DNA molecules in a defined way, in order to perform structural analysis of the B-S transition of DNA which may shed light on the interaction of DNA with the RecA protein, responsible for recombination in E.coli. For force-extension measurements on an ensemble of DNA molecules we have constructed a new force apparatus, whose development and characterization is pre-sented. Inside this device functionalized DNA has to be chemically grafted between to substrates for force-extension experiments.
First the efficiency of functionalizing DNA with specific adhesion molecules such as biotin, digoxigenin and thiol for end-grafting DNA onto a substrate is determined.
We show that at least about 70 % of a DNA assay can be labelled with biotin or digoxigenin. The grafting of DNA onto different functionalized surfaces was verified by fluorescence microscopy. For end-grafting DNA specifically onto a streptavidin-coated glass surface via the biotin linker we found a maximal DNA density of at least about 0.14 molecules/µm. The same maximal density is achieved for coupling DNA onto a gold surface with the thiol linker. However in case of digoxigenin maximal graft-ing density is reduced as much as a factor of 10 whith respect to biotin-streptavidin grafting. Based on the binding kinetics of biotin-labelled DNA molecules we also sug-gest a diffusion-controlled binding model and propose future studies on the influence of tether-length on ligand-receptor binding kinetics.
The characterization of the DNA carpet by confocal fluorescence microscopy sug-gested to perform optical single-molecule experiments. The contour length of YOYO-1 stained λ-DNA at a dye:basepair ratio of 1 : 5 was measured to be 19.8. µm by stretching the DNA in an electric field and applying the WLC-model. The value is consistent with values available in literature. By measuring static and and dynamic properties of DNA molecules stretched by an electric field we have shown how one
111
could investigate the influence of hydrodynamic interactions in the case of electro-kinetic stretching of DNA molecules. Using the longitudinal resolution capabilities of confocal microscopy we measured for the first time 3-dimensional monomer density profiles of end-grafted DNA molecules of different length and found an excellent agree-ment with theoretical predictions. Using fluorescently stained colloids we labelled the end segment of individual DNA molecules and were able to measure the distribu-tion funcdistribu-tion of the end-segment of end-grafted DNA molecules. Our results provide the first direct experimental test of theoretical predictions for the conformation of end-attached polymers in the mushroom regime.
List of suppliers
Nucleic acids:
• Oligomers: MWG Biotech AG, Munich, and Thermo Electron Gmbh, Ulm
• λ-DNA: Fermentas GmbH, St. Leon-Rot and New England Biolabs GmbH, Frankfurt am Main
• KiloBase DNA marker: Amersham Bioscience Gmbh, Freiburg
• λ-DNA HindIII Digest: Amersham Bioscience Gmbh, Freiburg Antibodies and Enzymes:
• Strepatavidin: Roche Diagnostics Gmbh, Mannheim
• anti-digoxigenin: Roche Diagnostics Gmbh, Mannheim
• SfoI, ApaI, KasI, NaeI, AscI:New England Biolabs GmbH, Frankfurt am Main
• T4 Ligase:New England Biolabs GmbH, Frankfurt am Main
• T4 Polynucleoide Kinase: New England Biolabs GmbH, Frankfurt am Main
• Klenove Polymerase: New England Biolabs GmbH, Frankfurt am Main Dyes:
• YOYO-1, BOBO-1: Molecular Probes, Leiden (Distributor: MobiTec, G¨ottingen) 113
• Ethidium bromide: Amersham Bioscience Gmbh, Freiburg Beads
• Dynal-beads M-280 Streptavidin: Dynal Biotech Gmbh, Hamburg
• Magnetic anti-digoxigenin particles: Roche Diagnostics, Gmbh, Mannheim
• Fluospheres
• TransFluo Spheres NeutrAvidin: Molecular Probes, Leiden (Distributor: Mo-biTec, G¨ottingen)
• TransFluoSpheres unlabelled : Molecular Probes, Leiden (Distributor: Mo-biTec, G¨ottingen)
Miscellaneous:
• Gel Extraction Kit: Quiagen Gmbh, Hilden
• Ethanol: Sigma-Aldrich Chemie Gmbh, Steinheim
• 3-Amino-propyltriethoxysilane: Sigma-Aldrich Chemie Gmbh, Steinheim
• Glutaraldehyde: Polysciences, Warrington, USA
• DTT: Roche Diagnostics Gmbh, Mannheim
• Catalase: Sigma-Aldrich Chemie Gmbh, Steinheim
• Glucose Oxidase: Sigma-Aldrich Chemie Gmbh, Steinheim
• Surcose: Sigma-Aldrich Chemie Gmbh, Steinheim
• Tris base: Sigma-Aldrich Chemie Gmbh, Steinheim
• EDTA: Sigma-Aldrich Chemie Gmbh, Steinheim
• Agarose: USB, Cleveland
• TBE:Sigma-Aldrich Chemie Gmbh, Steinheim
• Nick Columns: Amersham Bioscience Gmbh, Freiburg
• Coupling buffer: Ademtech, 33600 Pessac France
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RecA and its complex with ADP-AIF: implication for decreased ATPase
RecA and its complex with ADP-AIF: implication for decreased ATPase