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

Abstract

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.

Figure 1

Chemical structure of 2′F-ANA compared with RNA and ANA.

Figure 2

(A) Quantitative real-time PCR (QRT-PCR) assay performed on RNA isolated from K562 cells transfected with PS-2′F-ANA and PS-DNA molecules. Data are presented as a function of c-MYB mRNA copies relative to GAPDH mRNA. Mock (control) cells were subjected to nucleoporation, but in the absence of DNA. (B) c-Myb protein western blot analysis performed on cell lysate obtained from K562 cells transfected with PS-2′F-ANA and PS-DNA molecules. Real-time PCR and western blot were performed 24 h post nucleofection.

Figure 3

(A) QRT-PCR assay performed on RNA isolated from K562 cells transfected with 2′F-ANA and PS-DNA molecules. Data are presented as a function of c-MYB mRNA copies relative to GAPDH RNA. Mock (control) cells were subjected to nucleoporation, but in the absence of ODN. MOH10 antisense is designed to target c-MYB mRNA region slightly shifted toward the 3′ end compared with the MOH1 and MOH2 sequences. (B) c-Myb protein western blot analysis performed on cell lysate obtained from K562 cells transfected with 2′F-ANA and PS-DNA molecules. Real-time PCR and western blot were performed 24 h post nucleofection.

Figure 4

QRT-PCR and corresponding western blot analysis of c-MYB expression performed (A) 72 h (B) 96 h and (C) 120 h post nucleofection.

Figure 4

QRT-PCR and corresponding western blot analysis of c-MYB expression performed (A) 72 h (B) 96 h and (C) 120 h post nucleofection.

Figure 5

(A) QRT-PCR and western blot analysis performed 24 h post nucleofection of cells with 1 µg of indicated ON in K562 cells. (B) QRT-PCR of c-MYB expression in K562 cells 24 h after nucleofection of ON in amounts indicated. (C) c-MYB expression in normal human CD34⁺ marrow cells 24 h post nucleofection with 1 µg of ON. Owing to the difficulty in obtaining CD34⁺ cells for this particular experiment, scrambled control for PS-DNA not tested.

Figure 6

Quantification of ODN delivered to cells by nucleoporation. (A) Slot blot of ON obtained from K562 cell lysate supernatants, and residual cell pellet 5 min, as well as 24, 72 and 96 h post nucleoporation. Upper rows are PS-DNA and PS-2′F-ANA–DNA (MOH10) antisense standards applied to membrane for quantification purposes. After transfer to the membrane, the blots were probed with a radiolabeled complementary sequence. Columns show material from untreated control cells (control), and cells nucleoporated with PS-DNA and PS-2′F-ANA–DNA chimera (MOH10). (B) Densitometry analysis of slot blot containing ON present in cell lysates, and remaining pellets, 5 min and 24, 72 and 96 h post nucleoporation of material. Control cells were nucleoporated in the absence of exogenous ON.