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PubMed2006

2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing.

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a >90% knockdown of c-MYB mRNA and protein expression, but the PS-2'F-ANA-DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2'F-ANA-DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.

Arabinonucleotides↗

PubMed2004

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Cell Division↗