G50 sephadex

Transcription templates were synthesized by PCR (GOTAQ G2, Promega) using a hybrid T7promoter/sequence-specific forward primer and a sequence-specific reverse primer. The PCR templates were either the WT minigene or the mutated ones. PCR products were checked on native agarose gel and purified on columns (DC5, Zymoresearch). Fluorescent RNAs were transcribed (37°C, 3 hours) in [40 mmol/L TRIS-Cl (pH7.9), 10 mmol/L NaCl, 6 mmol/L MgCl₂, 2 mmol/L spermidine, 5 mmol/L DTT, rNTPs 0.5 mmol/L each, Cy3-UTP 0.1 mmol/L (Jena Bioscience), RNAsin (1 U/μL, Promega), T7 RNA polymerase (1.25 U/μL), DNA template (12.5 ng/μL)]. RNAs were purified on G50 sephadex (GE Healthcare) and controlled by denaturing electrophoresis and fluorescent detection (Typhoon FLA 9500). Cy3 fluorescence and RNA absorbance were quantified on a De Novix DS-11 spectro/fluorimeter.

Gel exclusion chromatography was performed on a 40 cm (32 mL) G-50 Sephadex (GE Healthcare Life Sciences, Baie d’Urfe, QC) column in a buffer of 100 mM KPi, pH 7.0. The column was loaded with 2 mL of 10 mg/mL RNase and 35 fractions of 1 mL were collected. The fractions with the highest absorbance at 280 nm were pooled. The pooled fractions were then dialyzed into 10 mM sodium acetate buffer, pH 5.5. The dialyzed RNase A was further purified by ion exchange chromatography on SP Sepharose Fast Flow (GE Healthcare Life Sciences, Baie d’Urfe, QC), a strong cation exchanger, as per manufacturer's instructions. The salt gradient used was 0–0.4 M KCl. There were 60 fractions of 1 mL collected and the fractions with the highest absorbance at 280 nm were pooled again and used for nanopore experiments. The protein concentration of the pooled fractions was determined by measuring the absorbance at 280 nm and using the molar absorption coefficient (ε) of 9800 M⁻¹•cm⁻¹ for RNase A [54] (link). The calculated concentration was 101.5 µM or 1.39 mg/mL. 30–60 uL of this solution was used for nanopore analysis.