title: Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides.
abstract: Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase H degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC(50) values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS-[2'meRNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.
authors.affiliation: Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montreal, Quebec PQ, Canada H3A 2K6.
authors.forename: Chun-Nam
authors.initials: CN
authors.lastname: Lok
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Viazovkina
authors.forename: Kyung-Lyum
authors.initials: KL
authors.lastname: Min
authors.forename: Eva
authors.initials: E
authors.lastname: Nagy
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wilds
authors.forename: Masad J
authors.initials: MJ
authors.lastname: Damha
authors.forename: Michael A
authors.initials: MA
authors.lastname: Parniak
mesh_terms: D001089:Arabinose / Q000031:analogs & derivatives* / Q000737:chemistry*; D001483:Base Sequence; D002942:Circular Dichroism; D003855:Deoxyribose / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D008156:Luciferases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D016914:Ribonuclease H / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C118537:2'-deoxy-2'-fluoroarabinose; D016376:Oligonucleotides, Antisense; D003855:Deoxyribose; D001089:Arabinose; D008156:Luciferases; D016914:Ribonuclease H; C078856:ribonuclease HI