Ied RNA. The strong assistance was treated with MeNH2 in EtOH (33 , 0.five mL) and MeNH2 in water (40 , 0.five mL) for 7 h at room temperature. (For RNA containing 5-aminoallyl uridines, the column was initially treated with ten diethylamine in acetonitrile (20 mL), washed with acetonitrile (20 mL) and dried. Then, the solid help was treated with MeNH2 in EtOH (33 , 1 mL) and NH3 in H2O (28 , 1 mL) for ten min at room temperature and 20 min at 65 .) The IGF-1R Accession supernatant was removed from and the strong support was washed 3 occasions with ethanol/water (1/1, v/v). The supernatant and the washings were combined with all the deprotection solution in the residue and the entire mixture was evaporated to dryness. To take away the 2-silyl guarding groups, the resulting residue was treated with tetrabutylammonium fluoride trihydrate (TBAF3H2O) in THF (1 M, 1 mL) at 37 overnight. The reaction was quenched by the addition of triethylammonium acetate (TEAA) (1 M, pH 7.four, 1 mL). The volume of the option was reduced plus the remedy was desalted with a size exclusion column (GE Healthcare, HiPrep 26/10 Desalting; 2.6 10 cm; Sephadex G25) eluating with H2O; the collected fraction was evaporated to dryness and dissolved in 1 mL H2O. Evaluation of your crude RNA following deprotection was performed by anionexchange chromatography on a Dionex DNAPac PA-100 column (4 mm 250 mm) at 80 . Flow rate: 1 mL/min, eluant A: 25 mM Tris Cl (pH eight.0), 6 M urea; eluant B: 25 mM Tris Cl (pH eight.0), 0.5 M NaClO4, six M urea; gradient: 0- 60 B in a within 45 min or 0-40 B in 30 min for brief sequences as much as 15 nucleotides, UV-detection at 260 nm. Purification of 2-O-(2-Azidoethyl) Modified RNA. Crude RNA items were purified on a semipreparative Dionex DNAPac PA-100 column (9 mm 250 mm) at 80 with flow rate 2 mL/min. Fractions containing RNA were loaded on a C18 SepPak Plus cartridge (Waters/Millipore), washed with 0.1-0.15 M (Et3NH)+HCO3-, H2O and eluted with H2O/CH3CN (1/1). RNA containing fractions were lyophilized. Evaluation of the quality of purified RNA was performed by anion-exchange chromatography with identical situations as for crude RNA; the molecular weight was confirmed by LC-ESI mass spectrometry. Yield determination was performed by UV photometrical evaluation of oligonucleotide solutions. Mass Spectrometry of 2-O-(2-Azidoethyl) Modified RNA. All experiments were performed on a Finnigan LCQ Advantage MAX ion trap instrumentation Glucosidase medchemexpress connected to an Amersham Ettan micro LC technique. RNA sequences wereArticleanalyzed within the negative-ion mode with a possible of -4 kV applied towards the spray needle. LC: Sample (200 pmol RNA dissolved in 30 L of 20 mM EDTA answer; typical injection volume: 30 L); column (Waters XTerraMS, C18 2.5 m; 1.0 50 mm) at 21 ; flow rate: 30 L/min; eluant A: 8.6 mM TEA, 100 mM 1,1,1,3,three,3-hexafluoroisopropanol in H2O (pH eight.0); eluant B: methanol; gradient: 0-100 B inside a inside 30 min; UV-detection at 254 nm. Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Labeling. 2-O-(2-Azidoethyl) modified RNA (60 nmol) was lyophilized within a 1 mL Eppendorf tube. Then, aqueous solutions of F545 (Acetylene-Fluor 545, Click Chemistry Tools), CuSO4, and sodium ascorbate have been added consecutively; acetonitrile was added as cosolvent36 to reach final concentrations of 1 mM RNA, two mM dye, 5 mM CuSO4, 10 mM sodium ascorbate, along with a H2O/acetonitrile ratio of 4/1 in a total reaction volume of 60 L. The reaction mixture was degassed and stirred for 3 to 4 h under argon atmosphere at 50 . To monit.
