Secondary Structure Analysis of Intra- and Intermolecular Guanine Quadruplexes and Targeting through Complementary and Homologous Binding using Peptide Nucleic Acid

Abstract

Many of the projects discussed in this thesis utilize one or more of the binding modes shown in Figure 1.12. As a result of this work, we expand our understanding of GQ secondary structures and show the ability to target these GQs using multiple binding modes. Chapter 2 analyzes variations on a short peptide nucleic acid (PNA) probe that homologously binds to guanine-rich telomeric DNA. We compare the PNA probe with and without backbone modifications and utilize a GQ binding small molecule fluorophore; a combination of two binding modes. This work identifies that TO-labeled probes show significantly increased thermal stability compared to their unlabeled counterparts. Modifications inducing a right handed helix in the PNA show no difference in thermal stability. This short probe does not possess selectivity as evidenced by in vitro translation experiments performed in cell lysate. In Chapter 3, we expand the current literature on RNA:DNA heteroquadruplexes (RDQ). This GQ contains both RNA and DNA guanine tracts and has recently been identified as a regulator between transcription and translation. We show the formation and stabilities of three suspected RDQ sequences using an optimized scaffold duplex. The formation of the RDQs was also monitored with a small molecule fluorophore, Thioflavin T.Chapter 4 utilizes the RDQ systems created in Chapter 3 and compares them to DNA and RNA homoquadruplexes. With this chapter, we make DNA:DNA and RNA:RNA homoquadruplexes using the scaffold duplex created in chapter 3 and compare their biophysical properties to our RDQs. A modified PNA oligomer is then used to duplex invade each heteroquadruplex. We identify that RDQs possess characteristics of both RNA and DNA heteroquadruplexes. Our PNA probe is also able to sufficiently target each heteroquadruplex with varying IC50 values. Chapter 5 describes the use of dual gammaPNA (γPNA) probes that hybridize to alternating sites along a telomere. Each probe contains a fluorophore, and can undergo Forster resonance energy transfer (FRET). We study the biophysical characteristics of these probes and observe bright staining of telomeres in nuclei. Finally, we show the incorporation of additional fluorophores does not significantly improve FRET efficiency.