Improvements in siRNA properties mediated by 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)

Abstract

RNA interference (RNAi) has emerged recently as an efficient mechanism for specific gene silencing. Short double-stranded small interfering RNAs (siRNAs) are now widely used for cellular or drug target validation; however, their use for silencing clinically relevant genes in a therapeutic setting remains problematic because of their unfavourable metabolic stability and pharmacokinetic properties. To address some of these concerns, we have investigated the properties of siRNA modified with 2'-deoxy-2'-fluoro-beta-d-arabinonucleotide units (araF-N or FANA units). Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA. We also show that the incorporation of FANA units into siRNA duplexes increases activity and substantially enhances serum stability of the siRNA. A fully modified sense 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA) strand when hybridized to an antisense RNA (i.e. FANA/RNA hybrid) was shown to be 4-fold more potent and had longer half-life in serum (approximately 6 h) compared with an unmodified siRNA (<15 min). While incorporation of FANA units is well tolerated throughout the sense strand of the duplex, modifications can also be included at the 5' or 3' ends of the antisense strand, in striking contrast to other commonly used chemical modifications. Taken together, these results offer preliminary evidence of the therapeutic potential of FANA modified siRNAs.

Figure 1

Chemical structures of FANA and RNA.

Figure 2

Efficacy of the different siRNAs at inhibiting luciferase in HeLa X1/5 cells. Cells were transfected with 60 nM siRNA with modifications in the sense strand only (A), in the antisense strand only (B) or in both sense and antisense strands (C). Luciferase activity levels were measured 24 h post-transfection and normalized to metabolic activity. The normalized luciferase activity was then determined as a percentage of luciferase activity as compared with an unrelevant control siRNA (siRNA-CTL) set at 100%. Data represent mean normalized luciferase activity ± SEM. Luciferase mRNA levels were quantified by real-time PCR analysis (relative to expression of the house keeping gene GAPDH) 24 h post-transfection. Bars show mean Luciferase/GAPDH ratios ± SEM.

Figure 3

Potency of FANA-containing siRNA at inhibiting the luciferase activity. Dose-responses were obtained for each siRNA by transfecting cells with different amounts of active siRNA for 24 h. Luciferase activity was measured and values normalized to the metabolic activity and compared with a control siRNA (siRNA-CTL) set at 100%. Data represent mean normalized luciferase activity ± SEM.

Figure 4

Duration of activity of modified siRNA. Cells were transfected with 60 nM siRNA. Luciferase activity was measured 4, 8, 24, 48, 72 and 96 h post-transfection. Data represent mean normalized luciferase activity ± SEM compared with a control siRNA (siRNA-CTL) set at 100%.

Figure 5

Serum stability of FANA-containing siRNA. The different siRNAs were incubated in 10% FBS at 37°C and aliquots were taken at the time points indicated. (A) The siRNA were separated by PAGE and visualized with SYBR gold. ‘ds’ depicts double-stranded siRNA marker and ‘ss’ single-stranded. (B) Bands were quantified by densitometry and the percentage of intact siRNA from initial amount set at 100%. Data represent mean values from three independent experiments ± SEM.