Factors governing the design, selection and cleavage of sugar-modified duplexes by ribonuclease H

The antisense principle bases its premise in the exquisite complementarity of a synthetic, chemically-modified oligonucleotide to tightly bind with a unique target RNA sequence. Rapid and selective genetic discrimination, as driven by the formation of multiple points of target contact, constitutes a central goal of oligonucleotide therapies. Most synthetic designs have, however, provided little structural insight on the role of the antisense oligonucleotide (AON) in triggering RNA cleavage of preformed hybrids, as catalyzed by a ubiquitous, intracellular enzyme known as ribonuclease H. The use of RNase H to assist AON inhibition of gene expression is crucial to mainstream antisense technologies, yet the precise mode by which this enzyme acts on AON/RNA duplexes remains unclear.; To address the role of substrate structure on enzyme activation, a dominant theme of this thesis highlights the design, synthesis and structural studies of novel AONs comprised of rigid 2'-deoxy-2'-fluoroarabino (2'F-ANA) or native (DNA) nucleotides, containing interspersed flexible (e.g. "2',3'-seconucleotides") or anucleosidic (e.g. butyl) residues. This unique AON class combines both pre-organization & flexibility within the hosting heteroduplex, which on their own usually prove detrimental towards enzyme trigger. Their combination, however, synergistically activates both E. coli and human RNases H, leading to potent destruction of duplexed RNA. These compounds thus represent the first examples of modified AONs lacking deoxyribose sugars that elicit RNase H activity comparably to the native (DNA) systems. DNA-derived AONs with acyclic residues also amplify enzyme-catalyzed target degradation, suggesting the added flexibility imparted to the substrate structure to be vital for ameliorating the protein/nucleic acid interaction. Melting and circular dichroic experiments have revealed that the enhanced dynamics associated with a particular acyclic modification remain globally undetectable, indicating the acyclic residues induce only local structural deformations to the helix architecture.; Intricate comparisons of the structural and biological properties of various acyclic residues (e.g. butyl, propyl and ethyl interresidue spacers) designed to locally compress or expand the AON helix backbone at a defined axial site has enabled a deeper understanding of the conformational factors that underlie the observed enhancements. (Abstract shortened by UMI.)...