Induction of RNase H activity by arabinose-peptide nucleic acids

Antisense oligonucleotides (AON) offer a rational approach for drug design. The specificity of AONs towards a complementary messenger RNA (mRNA) target via Watson and Crick base pairing as well as their ease of synthesis render this technology very attractive. RNase H-degradation of mRNA via formation of a AON/mRNA hybrid is crucial to mainstream antisense technologies. Numerous studies have demonstrated the importance of the AON's structure and conformational flexibility for efficient induction of RNase H activity. However the precise mode of action and substrate specificity of the RNase H are not fully understood at present. Our Laboratory recently discovered that incorporation of flexible acyclic linkers (e.g. butanediol, 2'-seco-RNA) significantly amplifies enzyme activity. Unfortunatly incorporation of such linkers was accompanied by a drop in the thermal stability of the AON/RNA hybrids. This prompted us to incorporate a less flexible linker such as a peptide nucleic acid, with the hope to maintain similar enzymatic activity while increasing the duplex thermal stability.;Incorporation of a PNA residue within DNA or 2'-FANA did not afford improvement in neither thermal stability nor enzymatic cleavage (except for homopolymeric sequences vs DNA) as compared to control or butyl-sequences.;This thesis highlights the synthesis of the 5'-amino nucleoside analogue required for the incorporation of the peptide nucleic acid in both 2'-fluoroarabinonucleic acid (2'F-ANA) and DNA. Circular dichroism experiments afforded information on the hybrid conformation in solution, whereas UV thermal melting studies provided a measure of the thermal stability of such hybrid duplexes. Finally, ability of various linker modified AON/RNA hybrids to activate the RNase H enzyme was evaluated in parallel with the corresponding native unmodified DNA/RNA hybrids.