Previously lyophilized modified sense or antisense RNA oligomers were reconstituted in distilled nuclease-free water (diH2O) to a concentration of 200 μM. To remove protecting trityl groups from the sense strand RNA oligomer, the strand was treated with tris(carboxyethyl)phosphine (TCEP; Sigma-Aldrich, St. Louis, MO) to obtain a 20 mM TCEP (Thermo Fisher Scientific, Pittsburgh, PA) and 0.1 M triethyl ammonium acetate (TEAA; Thermo Fisher Scientific) solution and a final RNA concentration of 100 μM. This mixture was placed in a thermocycler (Genius, Techne Ltd., Cambridge, UK) at 70°C for 3 min and subsequently returned to ambient temperature for 1 hour. The solution was then centrifuged in Microspin 6 desalting columns (Bio-Rad, Hercules, CA), according to the procedure delineated by the manufacturer, to remove the TCEP, TEAA, and protecting group salts. The concentration of RNA in solutions was then quantified using a NanoDrop (Thermo Fisher Scientific, Wilmington, DE). The two strand solutions were combined in equal single-oligomer mole proportion and diluted with phosphate-buffered saline (PBS) to afford a 50 μM solution of double-stranded siRNA. The resulting solution was heated to 70°C in the thermocycler for 3 min and was then removed from the thermocycler and allowed to cool to ambient temperature for 15 min. The annealed, reduced siRNA (annealed siRNA) solution was then frozen at −20°C, and siRNA concentration was quantified using the NanoDrop.

Polyacrylamide gel analysis was performed to confirm the annealing of the siRNA. Precast 15% acrylamide gels (Bio-Rad) were loaded with a solution containing 4 μl of annealed double-stranded siRNA or single-stranded RNA oligomers and 4 μl of loading buffer containing 50 mM tris-HCl, 5 mM EDTA, 25% glycerol, 0.2% bromophenol blue (Thermo Fisher Scientific), and 0.2% xylene cyanole FF (Sigma-Aldrich). These gels were run in tris-borate EDTA (TBE) buffer at 100 V for 90 min. The gels were stained with SYBR Gold (Thermo Fisher Scientific) according to the manufacturer’s instructions for 5 min with agitation. The gels were imaged with a ChemiDoc imaging system (Bio-Rad) (fig. S1A).

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