Molecular recognition with DNA nanoswitches

• Main Author: Mountford, Christopher Paul
• Published: University of Edinburgh 2008
• Subjects: 572.85
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659671

This thesis describes an investigation of the use of DNA junctions as functional nanoswitches, capable of molecular recognition tasks in nucleic acids. The topological branch point allows a conformational change from a planar, open structure to a closed conformation which allows the junction arms to stack. A stable nanoswitch was designed which formed a complete cruciform structure only when hybridised to a complementary target. Introduction of single base mismatches in the target at the nanoswitch branch point caused changes in FRET efficiency, depending upon the mismatch in question. This methodology was shown to be applicable to both DNA and RNA target sequences. The structural origins of these changes in FRET efficiency were then investigated using time-resolved fluorescence spectroscopy. Donor florescence decays were fitted using an assumption of a Gaussian distribution form for the fluorophore separation. The results showed a maximal change in peak position of 10 Angstroms, illustrating the sensitivity of these devices. The fluorescent analogue of adenine, 2-amino purine (2AP), was used as s direct probe of the branch point structure. Finally, a high throughput analysis mechanism was demonstrated. This used a fluorescence lifetime plate reader to obtain donor fluorescence decays for numerous samples in a short exposure time, which were used to determine the nanoswitch conformation. This method was initially shown to be applicable to DNA nanoswitches, with the use of FLIM also demonstrated, before the method was shown to be useful for distinguishing single base mismatches. Using a time-resolved approach has the benefit of being independent of local sample concentration, ideal for nanotechnology applications. These nanoswitches have been shown to be promising prototype molecular recognition devices.