Evaluation of the sequence-specific DNA hybridization and cleavage capabilities of peptide nucleic acid-titanium dioxide nanoconjugates

Organisms often acquire deleterious DNA mutations that cause cancer or infections which may negatively impact their health. More sensitive methods of imaging and treatment are needed to provide earlier detection and more specific removal of such deleterious DNA than current techniques. Previously, a DNA-TiO2 nanoconjugate was developed that is capable of hybridizing to target DNA intracellularly in a sequence-specific manner, with the ability to cleave DNA when excited by electromagnetic radiation. However, this DNA-TiO 2 nanoconjugate may be susceptible to degradation by nucleases and only able to hybridize transcriptionally active forms of DNA which may lower their intracellular targeting efficiency. The purpose of this dissertation was to evaluate the DNA hybridization capabilities of peptide nucleic acid (PNA)-titanium dioxide (TiO2) nanoconjugates and evaluate their capacities to induce DNA damage as a gene targeting agent. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes, which hybridize to single strand DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double strand DNA (dsDNA), and form different PNA-DNA complexes. This dissertation describes the synthesis of PNA-TiO2 nanoconjugates and several novel approaches, using real-time PCR dissociation analyses and gel electrophoresis, developed to investigate the hybridization behaviors of these nanoconjugates. PNA-TiO2 nanoconjugates are able to hybridize to target ssDNA and plasmid DNA under physiological-like ionic and temperature conditions; finally, when modified by the addition of optical imaging agents or peptides PNA-TiO2 nanoconjugates retain the same (or in some cases enhanced) hybridization capabilities. Detailed characterization of PNA-TiO 2 nanoconjugates shows that they possess unique hybridization and modification characteristics which set them apart from both PNAs and DNA-TiO2 nanoconjugates. The PNA-TiO2 nanoconjugates can be used for such in vitro experiments that can not be done with DNA-TiO2 nanoconjugates; their ability to invade plasmid DNA indicates that they may be beneficial both in intracellular and in in vivo studies. The capacity of PNA-TiO2 nanoconjugates to induce site-specific DNA damage is also evaluated. It is demonstrated that the ability of PNA-TiO 2 nanoconjugates to induce site-specific DNA damage is significantly enhanced by conjugation of the optical imaging agent alizarin red s and rationally designed PNA sequence.