Oligonucleotide Synthesis

Oligonucleotides were synthesized using modified (2ʹ-fluoro [2ʹ-F], 2ʹ-O-methyl [2ʹ-O-Me]) phosphoramidites with standard protecting groups (ChemGenes). Phosphoramidite solid-phase synthesis was done on a MerMade 12 (BioAutomation) and Dr. Oligo 48 (Biolytic) using modified protocols. Unconjugated oligonucleotides were synthesized on controlled pore glass (CPG) functionalized with a long-chain alkyl amine (LCAA) and unylinker terminus (ChemGenes). Cholesterol-conjugated oligonucleotides were grown with the cholesterol moiety bound to a tetraethylenglycol (TEG) attached through a succinate linker to LCAA-CPG support (ChemGenes). Phosphoramidites were prepared at 0.1 M in anhydrous acetonitrile (ACN), with added dry 15% dimethylformamide (DMF) in the 2ʹ-O-ME U amidite. 5-(benzylthio)-1H-tetrazole (BTT) was used as the activator at 0.25 M. Detritylations were performed using 3% trichloroacetic acid in dichloromethane (DCM). Capping was done with nontetrahydrofuran-containing reagents CAP A, 20% n-methylimidazole in ACN; and CAP B, 20% acetic anhydride (Ac2O), 30% 2,6-lutidine in ACN (synthesis reagents were purchased at American International Chemical [AIC]). Sulfurization was performed with 0.1 M solution of 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine (ChemGenes) for 3 min. Phosphoramidite coupling times were 3 min for all amidites used.