Antiviral APOBEC3 genes: Expression, regulation and association with clinical markers

Antisense oligonucleotides are valuable research tools that can be use to control gene expression and identify potential therapeutic targets. In addition, antisense oligonucleotides have the potential for use as therapeutic agents. The research presented in this thesis describes novel antisense cleavers, oligonucleotides that are designed to hydrolyze their RNA targets. Such antisense cleavers provide an alternative to antisense oligonucleotides whose mechanism of action relies on steric blocking or target cleavage by RNase H.;Two different types of antisense cleavers are described. The first type of cleaver targets bulged nucleotides formed when the cleaver oligonucleotide binds to its RNA target. Antisense molecules with imidazole or primary amines attached at an internal position and the 5'-end of the oligonucleotide were synthesized. These bifunctional antisense cleavers showed very modest hydrolytic activity (1-2% after 24 hrs at 37°C) when a single base bulge was targeted. Significantly better hydrolysis (52% after 48 hrs at 37°C) was observed when a two base bulge was targeted with a cleaving group consisting of two imidazole groups tethered at the 2'-position of an internal nucleotide of the antisense molecule. Structural and experimental data suggests that a two base bulge forms the proper orientation for strand hydrolysis by inline attack on the phosphate. The increased hydrolysis of the target in this conformation confirms this data and shows that targeting a two base bulge is effective in the design of internally conjugated antisense cleavers.;Because a number of different functional groups could contribute to cleaver activity, an unbiased combinatorial synthetic approach employing peptide libraries was used to identify active cleavers. The peptide library is conjugated to the 2' position of an internal residue in the antisense oligonucleotide to target a two base bulge in the RNA target. A novel activity assay was developed to screen for active RNA cleavers in the libraries. A second identification assay was employed to collect active cleavers and identify their composition by mass spectrometry. Using this approach an active cleaver with the peptide composition CR2K was identified.;The second type of antisense cleaver contains a single peptide conjugated through the epsilon-amino group of a lysine residue to the 5' end of an antisense oligonucleotide. A simplified support based method was used to prepare KHK, HKH, and HHKHH peptide-conjugated antisense cleavers. All three conjugates were shown to have RNA cleaving activity in vitro assays. Conjugate HHKHH-14 had the highest observed rate with a t 1/2 of ∼16 hrs, which compares favorably to other types of antisense cleavers reported in the literature.;Unlike chimeric oligonucleotides, which require RNase H for activity, or RNAi, antisense cleavers that use cleaving moieties directly conjugated to the oligonucleotide do not require additional cellular factors for their activity. The experiments described in this thesis show that peptide-oligonucleotide conjugates can serve as antisense cleavers. The synthetic methods and screening assays presented here should prove useful in identifying peptides that when conjugated to antisense oligonucleotides enable the conjugate to efficiently hydrolyze its RNA target. Such molecules could broaden the applications of antisense oligonucleotides as research tools and therapeutic agents.