Binding of Gamma PNA to Targets Derived from Viral and Human Genomes

The targeting ability of peptide nucleic acids (PNAs) to functional sequences in the genome is crucial for therapeutic development. While many scientific reports have been published on PNAs, the binding ability of newer generation gamma substituted PNAs (yPNAs) to different target sequences is yet to be evaluated. Towards this objective, we have studied the interactions of complementary yPNAs to canonical and non-canonical structures of nucleic acid targets (DNA/RNA) that are modeled based on viral or human regulatory sequences.

Chapter 1 summarizes a vast number of existing literature on various applications of the first generation unmodified PNAs in gene targeting and discusses different backbone modified new generation PNAs that have advantages over the first generation. Chapter 2 characterizes guanine quadruplex (GQ) formation by a guanine-rich conserved sequence in the NS5 protein-coding region of the West Nile virus (WNV) genome at physiological temperature and potassium concentration. We further applied various spectroscopic techniques to confirm the invasion of the GQ structure by complementary yPNAs. The yPNAs showed very strong binding to the target with low fM affinity at physiological temperature and potassium concentration. Chapter 3 focuses on another DNA secondary structure called intercalated motif (i-motif) formed by the cytosine-rich sequence at the KRAS promoter. This chapter first characterizes the i-motif formation at acidic and neutral pH followed by the successful invasion of the i-motif structure by unmodified and analogous y-modified complementary PNAs. We also observed the formation of intermolecular i-motifs by the overhangs of the DNA-yPNA heteroduplexes. Lastly, chapter 4 focuses on developing mitochondria penetrating PNA-Peptide and yPNA-Peptide conjugates, which can penetrate the cell membrane and mitochondrial double membrane and invade duplex mitochondrial DNA. The chapter demonstrates synthesis and optimizations of mitochondria penetrating PNA-Peptide and yPNA-Peptide conjugates. We have also presented the mitochondrial localization data for some of the PNA-Peptide and yPNA-Peptide conjugates. Finally, chapter 5 discusses the prospect of yPNA-based gene targeting in the context of various therapeutic applications.