PUB - Publikationen an der Universität Bielefeld

Recreating DNA-protein interaction in vitro could unravel the relationship between their structure and function, which necessarily requires transforming the DNA from its coiled state to a linear form. In this work, two main techniques were developed to realize this aim namely, orientation-defined DNA alignment and realtime monitoring of polymerase movement. Orientation-defined alignment of DNA was performed by bi- functionalization of DNA with two different pairs of chemical groups namely, thiol and biotin or thiol and silane at the termini. The terminal functionalized DNA was dropped on a nano-fabricated electrode and aligned across the gold and silicon dioxide surface using electrokinetic force. The Movement of polymerase in realtime was monitored us- ing molecular-beacons as reporters. The molecular beacons bind at regular intervals on a DNA that contains highly repeated sequences called homopolymer DNA. The homopolymer DNA will act as a template for the polymerase. The total fluorescence intensity decreases as the polymerase copies the second strand of DNA, due to removal and closing of molecular beacons. E. coli DNA polymerase I, T7 DNA polymerase, T7 RNA polymerase, and N4 virion RNA polymerase were used to validate this new technique.