Development of Programmable Gamma Peptide Nucleic Acids (γPNAs)

The first part of this thesis describes the development of a particular class of γPNA monomers containing orthogonal protecting groups that enables chemical functionalization and diversification directly on resin. In addition to achieving tight and sequence-specific binding, it is also essential to be able to tailor-design the functional properties of oligonucleotide molecules towards specific applications, such as biocompatibility, cellular delivery, and pharmacokinetics for drug development. Such a capability would allow oligonucleotide probes and reagents to be synthesized and decorated with the desired chemical functionalities from a small, universal set of monomers as opposed to having to prepare a new set for each functional group—a common practice in solid-phase chemical synthesis. The second part is focused on addressing the challenge in targeting the secondary and tertiary structure of RNA through the development and evaluation of the binding behaviors of selfavoidance nucleobases. Instead of using high-affinity nucleic acid probes to target these complex RNA structures, which is necessary to overcome the thermodynamic barriers imposed by the intricate molecular architectures, but in the process runs the risk of indiscriminate binding, we explore an alternative strategy by targeting both RNA strands in the stem region. This is accomplished by application of γPNAs containing self-avoidance nucleic acid recognition elements. These are engineered nucleobases that are unable to hybridize with their partners but would maintain recognition specificity with RNA. Even though their binding affinity with RNA is significantly reduced as compared to that of γPNA and γPNA, it is compensated by the binding free energy gained from targeting both strands of the RNA duplex—a molecular architecture that seeds RNA folding. A distinct advantage of such a nucleic acid system is that in addition to being able to target structured RNA, they are selective for them over the unstructured, linear strands. Moreover, they maintain recognition specificity with RNA, a recognition feature that is difficult to achieve with tight-binding oligonucleotides. The third part of my thesis is concentrated on the design of cooperative-binding nucleic acid probes for targeting CAG-RNA triplet repeats associated with Huntington’s disease as a model system. In contrast to the traditional practice, where cooperative binding is often achieved through terminal base-stacking, along with other non-directional molecular interactions such as π-π, electrostatic, and hydrophobic, which are difficult to control and fine-tune, we explore Watson-Crick base-pairing as a means to achieve cooperative binding between the adjacently bound probes on the RNA template. These probes are relatively small in size, six units in length, and contain two complementary arms. When the arms are strategically placed and the probes are optimized, they favor binding with the expanded RNA-triplet repeats over the normal-length wildtype. Although there is still a lot of work to be done, this research lays the foundation for the development of such oligonucleotide molecules, not only for targeting CAG-RNA repeats associated with Huntington’s disease but also for targeting other triplet-repeated sequences that are associated with a slew of neuromuscular and neurodegenerative diseases.