Mechanical Streching and Lightscattering on DNA

Abstract

The goal of the present work was to establish robust and reliable methods for the preparation and characterization of dense carpets of long-chain DNA attached with both ends simultaneously to the surfaces of a surface force apparatus. This provides the perspective of an experimental platform of studying strongly extended DNA with structure-sensitive methods such as X-ray scattering or optical birefringence. This approach should allow to elucidate the (so far unresolved) structural origin of the plateau in the force-extension curve observed in the single-molecule experiments, and to study the role of protein binding in large-scale structural changes in genomic DNA.<br /><br />In comparison to traditional single-molecule force experiments, the controlled stretching of a large ensemble of DNA molecules using a surface-force apparatus imposes stringent conditions to sample preparation: (i) the end-grafting of DNA to the substrate has to be strong enough allowing overstretching of the molecules, (ii) the end-grafting has to be specific for given substrate and finally (iii) in order to obtain measurable X-ray scattering or birefringence signals, the carpets, in addition to the above mentioned criteria, should ideally be prepared at densities at which the DNA forms brushes.<br /><br />In this work we have thus developed, on the one hand, an experimental route to the preparation of dense, strongly anchored DNA carpets suitable for surface force experiments, and, on the other hand, new methods to characterize tethering density and mechanical stability under external force.<br /><br />The mechanical stability of DNA-surface links has been improved by using long-chain DNA end-labeled with multiple biotins on one end and a thiol group on the other end.. This characterization was done by using direct observation of rupture events under external force in a confocal fluorescence microscope coupled to a simple extension device. We succeeded in characterization the mechanical stability of large ensembles of double-end-tethered DNA, clearly distinguishing single biotin from multiple biotin tethering. In addition we found that the end-modification of long-chain DNA with short oligonucleotides does not always provide the needed mechanical stability for stretching and that streptavdin (the biotin-binding protein) is not always strongly enough coupled to the substrate. This lead us to develop PCR (Polymerase Chain Reaction) synthesis of long-chain DNA end-labeled with multiple biotins on one end and a thiol group on the other end, The use of PCR, in principle, eliminates the problem of oligonucleotide ligation as the molecule is synthesized completely with its end-modifications. Additionally we have developed alternative end-grafting possibilities for long-chain DNA molecules so circumventing the problems found in streptavidin-surface grafting.<br /><br />Furthermore we have developed a new reversible combing method which indicates enhanced DNA tethering density. This method is based on unspecific electrostatic absorption of end-grafted DNA molecules to the surface which is previously elongated by a hydrodynamic flow. Through multiple combing of new DNA fractions, a systematic enhancement of the tethering density can be achieved, possibly allowing to produce DNA carpets at brush densities since the molecules can be released from the surface. Finally, evanescent wave quasi-elastic light scattering has been established as a sensitive tool for the detection of sub-monolayers of DNA tethered to solid substrates. This method might thus provide a new, marker-free method for the quantification of DNA tethering densities.