Oligonucleotides were synthesized by standard methods on solid supports using an Applied Biosystems 3400 DNA synthesizer. For thiolated strands, the 5′ end was modified with the Thiol Modifier C6 S-S phosphoramidite and standard protocols from Glen Research, Inc. DNA modified with Redmond Red on the 3′ terminus was prepared on Epoch Redmond Red CPG columns from Glen Research with ultramild phosphoramidites and reagents. For Nile Blue modified DNA, a 5-[3-acrylate NHS Ester]-deoxyuridine phosphoramidite (Glen Research) was incorporated at the 5′ terminus also using ultramild conditions. The DNA on solid support was then dried and reacted with a 10 mg/mL solution of Nile Blue perchlorate (Acros Organics) in 9:1 dichloromethane/N,N-diisopropylethylamine solution for approximately 24 hours. Excess reagents were then removed by washing three times each with dichloromethane, methanol, and acetonitrile.
Unmodified and thiolated oligonucleotides were cleaved from the solid support and deprotected by treating with concentrated ammonium hydroxide for 8 hours at 60°C. Redmond Red and Nile Blue modified DNA strands were cleaved from the support and deprotected according to ultramild conditions with 0.05 M potassium carbonate in methanol at ambient temperature for 8 hours.
Unmodified and thiolated oligonucleotides were cleaved from the solid support and deprotected by treating with concentrated ammonium hydroxide for 8 hours at 60°C. Redmond Red and Nile Blue modified DNA strands were cleaved from the support and deprotected according to ultramild conditions with 0.05 M potassium carbonate in methanol at ambient temperature for 8 hours.
