Structural Characterization of gamma-Modified PNAs and Invasion of MicroRNA

The research presented in this thesis describes an in depth analysis of gamma-modified peptide nucleic acid (gammaPNA) oligonucleotide analogs. This detailed analysis was performed in an attempt to determine the extent of steric accommodation for the side-chain modification at the gamma-backbone position within the PNA to improve conformation and hybridization properties. Along with these studies, a thorough structural characterization of these gamma-modified PNAs was completed to provide insights into the origin of gammaPNA preorganization. In addition to these analyses, gammaPNAs were also used as an antisense agent for the invasion of a secondary structured microRNA hairpin. The ability to bind to such structured RNA targets may provide a new path for the treatment of diseases resulting from misregulation of microRNAs;In order to achieve these goals, we first describe the modification of gammaPNAs with side-chains that are sterically hindered to varying degree. In these modifications, alanine, valine, isoleucine, and phenylalanine side-chains are incorporated at the gamma-backbone position of PNA thymine units. The resulting PNA oligomers, which contain a single modification, indicate conformational preorganization into a right handed helical structure. Further, they are able to bind to their DNA targets with better affinity than has been achieved with their unmodified counterparts.;We then performed an in depth structural characterization of the glycine PNA (unmodified), alanine, valine, and isoleucine gammaPNAs utilizing multidimensional and multinuclear NMR analyses. This was done to provide insight into the origin of the conformational preorganization. Results from these studies indicate that as a monomer unit the valine and isoleucine thymine monomers contained a preferred conformational structure; the alanine and glycine PNAs did not. Upon further analysis of the isoleucine thymine monomer along with reduced fragments of the monomer unit to ultimately contain only the side-chain and the backbone of the PNA, a preferred conformational structure still remained. These findings suggest that the side-chain along with the nitrogen in the backbone is sufficient to lock the compound into a preferred conformational structure.;The last part of this thesis describes the use of miniPEG modified gammaPNAs (MPgammaPNAs) as an antisense agent for the invasion of the structured secondary microRNA pre-let-7 target. The results presented provide evidence that the MPgammaPNAs are not only able to invade the pre-let-7 microRNA, but do so in a sequence-specific manner. Binding studies provide additional information on how well the gammaPNA invades the RNA structure. In addition, a detailed thermal denaturation study and other surface plasmon resonance studies provide further insight into how our developed system compares with other oligonucleotide analogs.;The gammaPNA results presented in this thesis may significantly impact the development and future design of antisense and antigene agents. Understanding the extent of modification that can be made at the gammaPNA backbone position as well as the origin of the preferred conformational preorganization will aid in making modifications to achieve improved binding affinity and sequence-specificity. The ability to successfully invade a structured RNA target provides further evidence of the potential applicability of the gammaPNAs for their development as therapeutic agents for the treatment of diseases.