Alliance chromatography system

C6 protected siRNA oligomers (IDT technologies) were reduced to their free thiol form using DL-Dithiothreitol (DTT) in 0.1M Triethylammonium bicarbonate (TEAbc) at pH 8.5 and then reacted with dithio-bis-maleimidoethane (DTME) in 300mM NaOAc/acetonitrile at pH 5.2. Purification of the siRNA-DTME intermediate by precipitation with ethanol was followed by reaction with the purified cyclic R11A peptide in 300mM NaOAc/acetonitrile at pH 5.2 with gentle mixing at room temperature. Analytical C-18 RP-HPLC purification of the resulting siRNA-DTME-LTP-Cy5.5 conjugates using triethylamine acetate (TEAA)/Acetonitrile gradients on a Waters Alliance chromatography system was followed by lyophilization and then re-lyophilization from nuclease free water. MALDI-TOF analysis of the purified conjugates on an Applied Biosystems Voyager workstation using 3-hydroxypicolinic acid (3-HPA) matrix in ammonium citrate allowed for confirmation of the expected mass and identity of the final siRNA-cyclic-LTP conjugate.

Preparation of samples for analytical evaluation of purity at the various synthesis intermediate steps involved dissolving each sample at a concentration of 1 mg/ml in 50%TFE/0.1%TFA and performing the separation on a Waters Alliance chromatography system using standard Acetonitrile-0.1% TFA gradient conditions at a flow rate of 1 mL/min.