Arabinonucleic Acids: 2′-Stereoisomeric Modulators of siRNA Activity

Abstract

We have investigated, for the first time, short interfering duplexes containing arabinonucleotides (ANA; the 2'-stereoisomer of RNA), as well as combinations of ANA with RNA, and their 2'-fluorinated derivatives 2F-ANA and/or 2'F-RNA. The results show that ANA is especially well accommodated in the sense strand of small interfering RNA (siRNA) duplexes, which can be extensively modified with little effect on potency. Furthermore, combining ANA with RNA and 2'F-ANA in siRNA passenger strands, particularly in patterns that bias duplex thermal stability, produces duplexes with similar (and sometimes enhanced) potency compared with native siRNA. Effective patterns of modification were identified against firefly luciferase screens in HeLa cells and then applied to knockdown of down-regulated in renal cell carcinoma (DRR), a novel and clinically tractable target for the treatment of glioblastoma.

Effect of Sugar 2′,4′-Modifications on Gene Silencing Activity of siRNA Duplexes

“…This protocol was adapted from Anzahaee et al [10]. In brief, DsRed DRR cells were seeded in a 6- or 24-well plate such that they would reach 60%–80% confluency at the time of transfection.…”

Structural properties and gene-silencing activity of chemically modified DNA–RNA hybrids with parallel orientation

“…iDD:R3, iDD:rF3), when carefully designed, can engage the RNAi pathway to knockdown the expression of a specific gene, with potencies similar to conventional aps -DNA:RNA and DNA:2′F-RNA duplexes. The parallel-stranded RNAi triggers studied here, like conventional siRNA duplexes, have the added advantage of using native RNA (or 2′F-RNA) as guide strands ( 38 , 39 , 41 , 47 , 48 ). Moreover, since the passenger strand of the ps -duplexes could be heavily modified (e.g.…”

Antibody-Antisense Oligonucleotide Conjugate Downregulates a Key Gene in Glioblastoma Stem Cells

“…All AONs were synthesized on an Applied Biosystems (ABI) 3400 DNA synthesizer at 1 μmol scale using Uny-linker CPG as solid support, except for the sense delivery oligonucleotide, which utilized cyanine 3 CPG as the solid support. The synthesis cycle conditions were as previously described, 14 with the exception that 0.02 M I 2 in 1:2:10 pyridine/water/tetrahydrofuran (THF) was used for the Cy3/DBCO-modified sense delivery oligonucleotides. When a phosphorothioate backbone was needed, a 0.10 M solution of ([dimethylamino-methylidene]amino)-3H-1,2,4-dithiozaoline-3-thione (DDTT) in Py:MeCN (9:1) was used for the oxidation step instead of the aqueous I 2 solution.…”

“…This protocol was adapted from Anzahaee et al. 14 Briefly, DRR+ GBM cells were seeded at 120,000 cells/well in a 6-well plate and allowed to adhere overnight. AONs were complexed to Lipofectamine 2000 (Thermo 11668027; at 3 μL/well) for 20 min in Opti-MEM media (Thermo 31985062) according to the Lipofectamine 2000 reagent protocol and added to the cells in DMEM media (11995605) for a final DMEM:Opti-MEM ratio of 1.5:0.5 and AON concentration of 25 nM.…”

“…It acts by regulating focal adhesion dynamics at the leading edge of migrating cells 12 and by activating AKT signaling, making it an ideal anti-invasion target. 12 , 13 , 14 …”