Abstract
Many different chemistries have been developed for the recognition of double-stranded DNA in a sequence specific manor, namely, gamma-peptide nucleic acids (γPNA), locked nucleic acids, Intercalating Nucleic acids, and Invader probes. While these DNA-targeting strategies have been well tested in separate model system no comparative study has been carried out evaluating their ability in the same model system. In chapter 2 I evaluated how these leading DNA-targeting approaches compare to each other in the same model system, giving valuable insight into probe chemistry selection for further biological applications. Further improvement of the binding capabilities of γPNA was developed in chapter 3 using chimeric probes. Since, intercalators are poorly tolerated in A-type duplexes, it was discovered that duplexes between γPNA and Invaders are easily denatured resulting in an adequate thermodynamic gradient for recognition of a DNA-target. This strategy resulted in improved DNA recognition from γPNA and improved mismatch discrimination. Since, γPNA chemistry is challenging to synthesize, in chapter 4 I evaluated Serine-γPNA (SγPNA) for DNA-recognition as an easier to synthesize alternative chemistry. I compared SγPNA with the corresponding Invader which also allowed for chimeric probes to be evaluated. SγPNA was successful at recognizing DNA with similar ability as Invader probes but had low solubility in G/C rich sequences and had poor mismatch discrimination of a singly mismatch target, additionally, some of the SγPNA sequences formed stable secondary structures reducing its DNA recognition ability. The use of chimeric probes resulted in a moderate improvement in recognition and specificity.
In chapter 5 and 6 I report projects which were less successful or are not fully completed yet. I designed Invaders to target a plasmid attempting to block a restriction enzyme from cutting its target site. Invader probes were demonstrated to have ability to bind to plasmid DNA but were unable to block the restriction enzyme from cutting the plasmid. Additionally, I explored LNA toehold design for DNA-targeting which, preliminary results show some DNA recognition ability. Further experiments will be needed to complete this project.