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.

Phosphorothioates, Essential Components of Therapeutic Oligonucleotides

“…Among others, 2¢-modifications can restore this high affinity, which is of course strongly desired. With the exception of 2¢ F-arabinonucleotides (FANA) [74], such modifications, however, are not compatible with activation of RNase H. A very interesting structural design succeeded in combining the two characteristics through the use of antisense gapmers (Fig. 11) [75].…”

2‘-Fluoroarabino- and Arabinonucleic Acid Show Different Conformations, Resulting in Deviating RNA Affinities and Processing of Their Heteroduplexes with RNA by RNase H,

“…Moreover, ANAs have nuclease resistance to serum and cellular nucleases that exceed those by DNA but do not match the stability seen with PS-DNA (12). Although PS-FANA AONs showed only relatively weak inhibition of cellular target expression, mixed-backbone oligomers with PS-DNA cores flanked by PS-FANA stretches were found to have potent antisense activity (15). So-called FANA/DNA altimers, consisting of alternating FANA and DNA residues (16), and FANA constructs containing acyclic nucleotide inserts (17) were also capable of promoting efficient cleavage of RNA targets by RNase H.…”

Antisense applications for biological control

“…Comparing the properties of ASOs based on phosphorothioated FANA (PS-FANA) and its GAPmer of the general structure PS-{FANA-DNA-FANA}, the latter was found to have exceptionally potent antisense activity. The antisense activities of the 2 0 -fluoroarabino GAPmer were sequence-specific and mediated by intracellular RNase H. Importantly, unlike 2 0 -O-methylribose chimeric compounds, the potencies of the 2 0 -fluoro-arabino chimeras were not limited by the length of the DNA core [Lok et al, 2002].…”