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.