Dnapac pa200

Oligonucleotides were prepared at final concentrations of 0.1 mg/ml in 50 mM Tris (pH 7.2), 10 mM MgCl₂ for assays in the presence of 3′-specific SVPD or in 50 mM sodium acetate (pH 6.5), 10 mM MgCl₂ for assays in the presence of 5′-specific PDE-II. The exonuclease (75 mU/ml SVPD or 500 mU/ml PDE-II) was added to oligonucleotide solution immediately prior to the first injection onto the HPLC column, and enzymatic degradation kinetics were monitored for 24 h at 25°C. Samples collected over 24 h were immediately injected directly onto a Dionex DNAPac PA200 analytical column at 30°C column temperature. The gradient was from 37% to 52% 1 M NaBr, 10% CH₃CN, 20 mM sodium phosphate buffer at pH 11 over 10 min with a flow rate of 1 ml/min. The full-length oligonucleotide amount was determined as the area under the curve of the peak detected at A₂₆₀. Percent full-length ON was calculated by dividing the area under the curve at a given time point by that at the first time point and multiplying by 100. Activity of enzyme was verified for each experiment by including a 20-mer oligodeoxythymidylate with a terminal PS linkage in each experiment. Each aliquot of enzyme was thawed just prior to the experiment. The half-life was determined by fitting to first-order kinetics. Each degradation experiment was performed in duplicate.

High-pressure
liquid-chromatography (HPLC) analytics were adapted from Kanavarioti^{65 (link)} and performed on an Agilent 1100 system (Agilent
Technologies, Inc., CA, USA). The column was a DNAPac PA200 (Thermo
Scientific, Waltham, MA, USA). A gradient for 16 min from 0% mobile
phase A (MPA_AEX; 10 mM NaOH, pH 12.0) to 95% mobile phase
B (MPB_AEX; 10 mM NaOH, 1.5 M NaCl, pH 12.0) at a flow rate
of 0.9 mL/min was applied followed by a re-equilibration to 100% MPA_AEX for 4 min. Before injecting the samples, a dilution with
MPA_AEX was done to stay within calibrated mass range.

20 mmol of purified ScMreB5^WT was denatured at 75°C. The denatured protein was spun at 21,000 g for 20 min at 4°C. Supernatant was filtered with 0.22-µm cellulose acetate filter (Corning), and the sample was loaded onto the pre-equilibrated (with buffer A, 2 mM Tris, pH 8) DNAPac PA200 ion-exchange column (Thermo Fisher Scientific). The runs were performed with a linear gradient of 0–20% buffer B (2 mM Tris, pH 8, and 1.25 M NaCl) for three column volumes, 20–40% buffer B for three column volumes, and 100% buffer B for two column volumes. 40 mmol of filtered solutions of ATP or ADP was used as a standard in the run. The absorbance at 255 nm was plotted against the conductivity for all the runs in Prism v5.00 for Windows.

RNA stability was tested in 80% rat serum over the course of 24 h and analyzed by anion-exchange chromatography under denaturing conditions. 30 μl of 200 μM miRNA in 80% rat serum (Wistar rats, male) were incubated in the autosampler of a Vanquish UHPLC (Thermo Fisher Scientific) at 37°C. Directly at t = 0, after 0.5, 4 and 24 h, 2 μl were injected onto a DNAPac PA200 analytical column (4 μm particle size, 4.6 × 150 mm, Thermo Fisher Scientific). The column temperature was set to 65°C. Samples were eluted with a gradient of buffer A (20 mM sodium phosphate, pH 11 (Sigma Aldrich)) and buffer B (20 mM sodium phosphate, 1 M sodium bromide (Sigma Aldrich), pH 11), going from 35% B to 98% B in 8 min, followed by a wash at 98% B over 1.5 min and column equilibration at a flow rate of 1 ml/min. UV detection was performed at 260 nm.