The Nature Of Intercalation And Detection Of Mismatches In DNA

Intercalation is an important binding mode for guest molecules to interactwith double-stranded DNA (dsDNA). Till now, it is still a big challenge toexperimentally investigate the intercalation interaction and the factors thatinfluence the interaction at a single molecule level. In this dissertation, bycombining atomic force microscopy (AFM)-based single-molecule forcespectroscopy (SMFS) and modification of AFM tip, we have realized the directmeasurement of the rupture force between pyrene and dsDNA at a singlemolecule level, which enables us to better understand the nature of intercalation.Moreover, by using rupture force as the signal, we have developed new methodsfor detection of mismatches in dsDNA at a single molecule level, as well as themulti-binding level.By using SMFS, we have contrastively studied the intercalations betweenpyrene and different types of dsDNA, including fully-matched dsDNA andmismatch-containing dsDNA. In order to measure the rupture force betweenpyrene and dsDNA directly, the pyrene moiety was covalently attached ontoAFM tip through a flexible poly(ethylene glycol)(PEG) chain, and the25-mersdsDNA was immobilized on gold substrate via a thiol-Au bond. Some criteriasuch as the profile of force-extension curve, the reasonable rupture extensions,single chain model fitting, and control experiments, are used to identify therupture events of pyrene-dsDNA complex. At a loading rate of5.0nN/s, therupture force for pyrene unbinding from fully-matched dsDNA is about54pN.In contrast, the rupture force for pyrene unbinding from mismatch-containingdsDNA is around37pN, and it is independent on the type of the mismatch, aswell as the number of mismatches contained in dsDNA. In the analysis of thedynamic force spectra, two transition barriers are revealed for pyrene unbindingfrom matched sites in dsDNA, and for pyrene unbinding from mismatch sites aswell. These results are indicative of a new method for detection of mismatchesin dsDNA by pyrene using SMFS at a single molecule level, even when only onemismatched base pair is included in dsDNA. Increasing the number of intercalation between pyrene and dsDNA, wehave enhanced the difference of rupture forces between pyrene modified AFMtip and the dsDNA modified surface, to distinguish the areas modified by themismatch-containing dsDNA from the ones modified by fully-matched dsDNAon the gold surface. Force maps with clear bands were obtained, while usingAFM-based force mapping to characterize the single type of dsDNA patternedgold surface, as well as two types of dsDNA patterned gold surface whichconstructed by microcontact printing method and self-assembly with chemicaladsorption. The AFM-based force mapping can serve as a new method to detectthe hybridization of nucleic acid at different areas on DNA microarrays.