Novel cross-linking technologies to assess protein-DNA binding and DNA-DNA complexes for gene delivery and expression.

摘要

The covalent crosslinking induced by a single-pulsed high-power laser takes place within several nano-seconds. This unique feature enables laser crosslinking to capture the freeze-frames of macromolecule interactions, thus making it well suited for kinetic study. In order to better understand the DNA-binding properties of topoisomerase II, the single-pulsed high-power laser was used to characterize the DNA-binding kinetics of yeast topoisomerase II in the absence of ATP. An equilibrium binding constant ({dollar}Ksb{lcub}eq{rcub}{dollar}) of {dollar}1.2pm 0.28times 10sp8 {lcub}rm M{rcub}sp{lcub}-1{rcub}{dollar} was determined from kinetic analysis. Combined with limited digestion with V8 protease and peptide microsequencing, the DNA-binding domain of yeast topo II was identified within a 29 kDa fragment.; To further evaluate the DNA-binding characteristics of topo II, a surface plasmon resonance (SPR) based biosensor technology, The BIAcore system, was applied to study real time kinetics. The human topo II DNA-binding constant ({dollar}Ksb{lcub}eq{rcub}{dollar}) of {dollar}7.9pm 0.29times 10sp7 {lcub}rm M{rcub}sp{lcub}-1{rcub}{dollar} was obtained. The effects of various clinically valuable anti-cancer drugs on topo II DNA-binding were also investigated.; Furthermore, a novel system, The biotin-avidin networked gene (BANG) system, was devised to crosslink DNA-DNA molecules via non-covalent interactions. This system exploits the stability of biotin-avidin interactions, which is one of the strongest known non-covalent interactions between protein and ligand, to form networks between different segments of DNA. The formation of such branched and networked DNA molecules was confirmed by electrophoresis, transmission and scanning electron microscopy. In vitro studies revealed that the system was stable over protease treatment and a wide range of temperatures, and also was accessible to proteins. In vivo characterization of the BANG system showed that the networks increased gene expression levels by at least 700% at the population level, and 2000% at individual cell level.; The potential of the BANG system is enormous. Besides the over-expression feature, the BANG system provides a new way to link DNA sequences together. Therefore, DNA networks can readily be formed with different constellations of DNA, which makes non-covalent cloning and expression possible.