Sep pakc18 cartridges

Oligonucleotides were either
purchased from Integrated DNA Technologies (Coralville, IA) or synthesized
in house on an Expedite 8909 solid-phase oligo synthesizer. Phosphoramidites
and reagents for the Expedite synthesizer were purchased from either
Glen Research (Sterling, VA) or Chemgenes (Wilmington, MA). Cleavage
of synthesized oligonucleotides from the solid support was performed
using 1 mL of AMA (1:1 mixture of 28% aqueous ammonium hydroxide and
40% aqueous methylamine) for 30 min at room temperature, while deprotection
was performed in the same solution for 20 min at 65 °C. Deprotected
oligos were lyophilized, resuspended in 100 μL of DMSO and 125
μL of TEA·3HF, and heated at 65 °C for 2.5 h to remove
TBDMS from 2′-hydroxyls. Following this deprotection, oligos
were purified by preparative 20% polyacrylamide gel electrophoresis
(19:1 with 7 M urea), desalted using Waters (Milford, MA) Sep-Pak
C18 cartridges, and characterized by high-resolution mass spectrometry
on an Agilent 6230 TOF mass spectrometer.

Oligonucleotides were either purchased from Integrated DNA Technologies or synthesized in-house on an Expedite 8909 solid-phase oligonucleotide synthesizer. Phosphoramidites and reagents for the Expedite synthesizer were purchased from either Glen Research or Chemgenes. Cleavage of synthesized oligonucleotides from the solid support was performed using 1 mL of AMA (1:1 mixture of 28% aqueous ammonium hydroxide and 40% aqueous methylamine) for 30 min at room temperature, while deprotection was done in the same solution for 20 min at 65 °C. Deprotected oligonucleotides were lyophilized, resuspended in 100 µL of dimethylsulfoxide (DMSO), and 125 µL of triethylamine (TEA) trihydrofluoride and heated at 65 °C for 2.5 h to remove tert-butyldimethylsilyl from 2′-hydroxyls. Following this deprotection, oligonucleotides were purified by preparative 20% polyacrylamide gel electrophoresis (19:1 with 7 M urea), desalted using Waters Sep-Pak C18 cartridges, and characterized by high-resolution mass spectrometry on an Agilent 6230 time-of-flight (TOF) mass spectrometer.