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. Author manuscript; available in PMC: 2015 Mar 30.
Published in final edited form as: Nat Biotechnol. 2014 Nov 17;32(12):1256–1261. doi: 10.1038/nbt.3078

Figure 2.

Figure 2

Biological activity of siRNNs. (a) Anti-Ago2 co-immunoprecipitation from cells transfected with 32P-labeled guide strand containing control wild-type charged phosphodiester (WT), six tBu-SATE or six control irreversible DMB phosphotriester oligonucleotides, duplexed to wild-type passenger strands and analyzed by denaturing gel electrophoresis. Note conversion of 32P-labeled tBu-SATE phosphotriester guide strand into wild-type phosphodiester guide strand. Input, 32P-labeled single guide strand. (b) 5′ RACE analysis of GFP mRNA from cells transfected with GFP tBu-SATE (18×) siRNN, control GFP siRNA and nontargeting control siRNA. The correct calculated cDNA RACE fragment size is 189 bp. Red arrow indicates correct Ago2 cleavage site. (c) Analysis of IFN-α induction in human PBMCs at 24 h post-treatment with highly stimulatory β-gal 2′-OH siRNA (2′ OH), 2′-modified siRNA (2′ mod) and O-SATE (14×) phosphotriester siRNN (2′ mod tBU-SATE). (d) Chemical determination of structures surrounding the thioester bond required for intracellular conversion by transfection of GFP siRNNs containing six phosphotriester groups on guide strand duplexed to wild-type passenger strands into GFP-expressing cells, followed by analysis for GFP RNAi responses by flow cytometry at 1–3 days. (e) Dose-dependent GFP RNAi responses by self-delivering GFP DD-siRNNA4 conjugates vs. nontargeting control DD-siRNNA4 conjugates analyzed for GFP RNAi responses by flow cytometry at 48 h. Error bar indicates s.d. (f) Dose-dependent GFP RNAi response comparison of self-delivering GFP siRNNs conjugated to two delivery domains (DD-siRNNA2), three delivery domains (DD-siRNNA3) or two location variations of four delivery domains (DD-siRNNA4) versus nontargeting control DD-siRNNA4 by flow cytometry at 48 h. (g) Dose-dependent cMyc RNAi response (1, 5, 25, 50, 100 nM) in MDA-MB-231-cMyc-HA breast cancer cells by self-delivering cMyc DD-siRNNA4 vs. nontargeting control DD-siRNNA4 (ctrl) conjugate (100 nM) at 48 h. (h) Treatment of U2OS osteosarcoma cells with self-delivering Plk1 DD-siRNNA4 conjugates induced Plk1 RNAi response and induced G2 phase cell cycle arrest, whereas nontargeting control DD-siRNNA4 (ctrl) conjugate did not.