Bonded phase t3 column

In vitro cA₄ cleavage assays were performed by incubating 2 μM Csm6 (wild-type) or Csm6 (H381A) mutant with 100 μM cA₄ in 150 μL reaction buffer containing 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, at 55 °C in a time course. At desired time points, the reaction was quenched by adding phenol-chloroform (sigma). The 100 μL deproteinized products were extracted, followed by adding 100 μL chloroform. Then the extracted products and controls were analyzed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corp. Milford, MA) equipped with a High Strength Silica (HSS)-based bonded phase T3 column, (2.1 × 100 mm, 1.8 um particle size), a Photo Diode Array (PDA) detector, and a SQD mass detector. Sample injections of 10 microliters each were used, and mass detection was performed in both positive and negative modes. Elution was performed with a linear gradient using the following method: eluent A (water with 0.05% TFA) and eluent B (acetonitrile with 0.05% TFA) at a flow rate of 0.3 ml/min as follows for an 8 min run: 0 to 5 min, 4 to 7 % B gradient; 5.1 to 6.6 min 95 % B to wash; and finally 6.7 to 8.0 min 4 % B to equilibrate. In vitro cA₄ cleavage assays were repeated at least three times, and representative results are shown.

The synthesis of cA_n was undertaken by incubating 1 μM Csm^crRNA binary complex containing Csm3^D36A mutant, 1.5 μM target RNA, 2.5 mM ATP and 5 mM MnCl₂ in the reaction buffer (20 mM Tris8.0, 100 mM NaCl, 100 mM KCl) in a 300 μl volume at 55 °C for 60 min. The reactions were quenched by adding 300 μL volume phenol-chloroform (Sigma). Then 200 μL deproteinized products were extracted, followed by adding 200 μL chloroform. The final extracted products and controls were analyzed using an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters Corp. Milford, MA) equipped with a High Strength Silica (HSS)-based bonded phase T3 column, (2.1 × 100 mm, 1.8 um particle size), a Photo Diode Array (PDA) detector, and a SQD mass detector. Sample injections of 10 microliters each were used, and mass detection was performed in both positive and negative modes. Elution was performed with a linear gradient using the following method: eluent A (water with 0.05% TFA) and eluent B (acetonitrile with 0.05% TFA) at a flow rate of 0.3 ml/min as follows for an 8 min run: 0 to 5 min, 4 to 7 % B gradient; 5.1 to 6.6 min 95 % B to wash; and finally 6.7 to 8.0 min 4 % B to equilibrate. The cAn synthesis assay was repeated at least three times, and representative results are shown.