Thermodynamic and kinetic investigation of nucleic acid molecular recognition by synthetic oligomers and small molecules

Research described in this thesis was focused on the elucidation of thermodynamic and kinetic parameters of the binding of locked nucleic acids (LNA) and peptide nucleic acids (PNA) to nucleic acid hairpins and G-quadruplexes. The studies described herein have provided information about the impact that secondary structure imposes on the hybridization of oligonucleotides. The interaction of small molecules interacalators and minor groove binders with unnatural nucleic acid duplexes such as PNA-DNA, LNA-DNA, LNA-RNA was also investigated.; To assess the impact of folding on their hybridization, biologically-inspired model hairpins have been designed and investigated. For example, the hybridization of antisense PNA probes that unsymmetrically target two 16-mer RNA hairpins inspired form the P9 loop of the wild type and a mutant DiGIR ribozyme was investigated. 11-mer PNAs have hybridized with high affinity and high hybridization rates to the RNA targets. Similarly, a panel of DNA dodecamer probes containing between 0-12 LNA modifications have been designed. These were targeted to an RNA hairpin secondary structure that is a model for a secondary structure element found in the trans-activating response element in the RNA from HIV-l. Each of the probes invaded the hairpin target and formed a stable hybrid duplex. Complex relationships between the thermal/thermodynamic stabilities of the duplexes and the number and placement of LNA modifications have been identified for both the hairpin and the unstructured RNA targets. The increase in stability per LNA modification relative to an unmodified DNA probe was significantly higher when targeting the hairpin versus the unstructured RNA, indicating that LNA modifications facilitate hybridization to folded target sequences.; Subsequently, a more complex RNA target folding, namely a G-quadruplexe structure has been studied. The target was a 37-mer RNA sequence previously selected from a combinatorial library for binding to the Fragile X mental retardation protein (FRMP), a protein believed to be involved in the transport of messenger RNA from the nucleus to the cytoplasm. Two PNA probes have been designed to invade RNA quadruplexes by different strategies: one was complementary to 7 nucleotides that are involved in the quadruplexes, while the other was homologous to the same site. The invasion of RNA quadruplex targets by both complementary and homologous PNAs has been demonstrated. The thermal stability of the PNA-RNA hybrids formed with the homologous PNA has increased by 20--26°C when compared with their correspondent targets. The results revealed the first example of a PNA 2-RNA hybrid quadruplex thus widening the scope of PNA recognition.; In a complementary direction, the recognition of unnatural nucleic acid duplexes such as PNA-DNA, LNA-DNA, LNA-RNA by small molecules was investigated. The effect of PNA-DNA duplex length on the templated aggregation of the cyamne dye DiSC2(5) and the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes have been explored. The results described in this thesis have implications for the design of effective antisense probes based on LNA and PNA. The work presented here has also potential applications in the design of efficient nucleic acid analogues as gene-targeted drugs, and components for nano-architectures and for the development of novel materials.