Oligo rp c 18 reverse phase column

Alkylated oligonucleotides were purified by semi-prep HPLC using an Agilent 1200 HPLC system and a Phenomenex Clarity Oligo-RP C-18 reverse phase Column (5 μm, 10 mm*250 mm). Buffer A: 0.1 M triethylammonium acetate pH 7.0; Buffer B: Acetonitrile. Flow Rate: 4 mL/min. The temperature was set to 45° C. The elution gradient was: t=0 to t=3 min: 6 to 10 % acetonitrile; t=3min to t=12 min: 10% acetonitrile; t=12 min to t=27 min: 10 to 13% acetonitrile; t=27 min to t=30 min: 13 to 50 % acetonitrile. Oligonucleotide containing fractions were collected manually. The elution times of the 4 oligonucleotides were as follow: non-alkylated 25-mer (19 min); non-alkylated 12-mer (21min); monoalkylated 25 mer (22 min); monoalkylated 12-mer (23 min). After lyophilisation, oligonucleotides were redissolved in water (3 mL) and further desalted by centrifugation using 3K Amicon centrifuge filters. In some cases, the monoalkylated oligonucleotides were further purified by page as described below for the purification of the crosslinked duplex. The yield of monoalkylated oligonucleotide was calculated by: %yield of monoalkylated oligonucleotide=100*(moles of monoalkylated oligonucleotide)/(mole of starting parent oligonucleotide). The yield of the 25-mer alkylated oligonucleotide was 0.044 μmol (11%; ± 2%) and the yield of the 12-mer alkylated oligonucleotide was 0.024 μmol (6 %; ± 1%).

The purity of all synthesized oligonucleotides was verified by using an Agilent 1200 HPLC system and Phenomenex Clarity Oligo-RP C-18 reverse phase Column (5 μm, 4.6 mm*250 mm). The elution system was the same as the one described above for the isolation of alkylated oligonuclotides. The temperature was set at 45°C. The elution times were as follow: non-alkylated 25-mer (30 min); non-alkylated 12-mer (33min); monoalkylated 25 mer (31 min); monoalkylated 12-mer (35 min); ICL (44 min).