DNA Strand Exchange on Liposome Surfaces

Abstract

The mechanism of DNA strand exchange, performed in vivo by proteins in the Rec A family, is despite extensive studies not understood in full detail. We therefore want to study the molecular parameters involved in the exchange using an in vitro platform. Positively charged liposomes have been widely used as non-viral gene-carriers, where the positive charges attract the negatively charged DNA. We here show that DNA strand exchange is significantly enhanced, both in rate and yield, on the surface of cationic liposomes.We study the strand exchange reaction by adding an excess of non-labeled single strands to liposome bound DNA duplexes labeled with a FRET pair and monitor the exchange as an increase in donor emission. The fastest exchange is seen for liposomes containing 35% cationic lipid and when the positive charges on the outer leaflet of the liposomes equal the amount of negative charges on the added DNA. Both increasing and decreasing the amount of DNA slows down the exchange rate, either due to competition for the binding sites or due to that the single-strands on average are further away from the duplexes. By studying the exchange of mismatched DNA we conclude that the DNA opens in a “zipper-like” manner on the liposome surface, since a mismatched base-pair in the end of the sequence affects the exchange rate much more than a mismatch base-pair in the middle of the sequence. The liposome surface can be easily functionalized with lipids bearing specific functionalities and we intend to modify the surface to study the effects of hydrogen bonding and/or hydrophobic interactions.