Abstract

Nucleic acids (NA) have emerged as a promising class of therapeutics with potential for the development of drug candidates aimed at treating genetic disorders. The clinical success of these platforms is attributable to several factors including their ease of synthesis, biocompatibility, and highly specific target binding via programmable hydrogen bonding patterns. Early investigation into the use of synthetic oligonucleotides (ONs) revealed that these molecules face a host of challenges when delivered in vivo, including susceptibility towards nucleases, rapid clearance, poor cellular uptake, and off-target effects. As a result, decades of research have focused on the development of methodologies to introduce chemical modification along the ON to enhance their therapeutic characteristics. However, no single ON design that addresses all these challenges has emerged, and as such, the development of potent chemical modifications is necessary for their continued success. Towards this end, here we focus on the development of new chemical modifications along the arabinonucleic acid (ANA) scaffold. These NA analogues have been shown to increase nuclease resistance and act as a substrate for RNase H1, an enzyme capable of cleaving the RNA strand of a DNA:RNA hybrid and the basis of the antisense approach. However, ANA forms duplexes of low thermal stability with their RNA targets relative to DNA and as such, in Chapters 2-3, we aimed to address these issues via installation of propynyl groups at the C5 position of pyrimidines using the phosphoramidite approach. These modifications improved the thermal stability of duplexes formed between ANA and target RNA when placed consecutively while reducing affinity for DNA targets in several ON systems. Moreover, modified ONs were compatible with E. coli RNase H mediated cleavage of RNA including in uniformly modified systems, while simultaneously enhancing nuclease stability. Furthermore, in Chapter 4 C5-propynyl-2'-deoxy-2'-fluoro analogs of ANA (FANA) were investigated in similar ON systems. Duplexes containing uracil analogs displayed greater stability of duplexes formed with RNA but not DNA targets and cytosine analogs were exceedingly stabilizing towards RNA targets. Similarly, all modified ONs were substrates for E. coli RNase H with rates comparable to DNA however, only minor improvements in nuclease stability were observed for these analogs. With the goal of exploring these modifications in vitro, we explored approaches to conjugate modified ONs to a ligand for enhanced cellular internalization. During our investigation, we developed a novel linker phosphoramidite containing a disulfide bond which would allow for reductive responsive release of the ON upon cellular internalization. In Chapter 5 we describe the synthesis of this linker, and how it can be used to incorporate several 5'- functional handles for bioconjugation. The results of this thesis contribute towards developing new chemical modalities which can be harnessed to tune the therapeutic properties of ONs for their continued success in the clinic.