Deprotection and Purification of Oligonucleotides

All oligonucleotides were cleaved and deprotected using ammonium hydroxide and 40% aqueous (aq.) methylamine (AMA) in a 1:1 ratio for 2 h at room temperature. The oligonucleotide solutions were then filtered to remove the CPG from the cleaved oligo. The filtrate was then cooled with dry ice and then dried under vacuum in a SpeedVac. The resulting pellets were resuspended in 5% ACN in water. The purification of the unconjugated strands were performed on an Agilent 1200 system, equipped with a Source 15Q anion exchange resin (GE Healthcare; 10 × 100 mm custom-packed column), using the following conditions: eluent A, 20% ACN, 20  mM sodium acetate (pH 5); eluent B, 1 M sodium perchlorate in 20% ACN; gradient, 0% B 2 min, 35% B 12  min, clean, and re-equilibration to initial conditions 6 min. Purification of cholesterol-conjugated strands was performed on the same equipment but equipped with a PRP-C18 (Hamilton), a polymer reverse-phase column (10 × 100 mm), using the following conditions: eluent A, 50 mM sodium acetate in 5% ACN; eluent B, ACN; gradient, 0% B 2 min, 0%–40% B 1 min, 40%–70% B 9 min, clean, and re-equilibration 6 min. Temperature 70 °C and flow rate 40 mL/min were the same in both cases. Peaks were monitored at 260 nm. The pure oligonucleotide fractions were collected, individually characterized by liquid chromatography-mass spectrometry (LC-MS), combined, frozen, and dried in a SpeedVac overnight. Oligonucleotides were resuspended in 5% ACN and desalted through fine Sephadex G-25 (GE Healthcare; 10 × 200 mm custom-packed column) and lyophilized. All reagents mentioned above were purchased from Sigma-Aldrich and used as per the manufacturer’s instructions, unless otherwise stated.