Template attachment and transcription

The template preparation and transcription were performed according to the method described previously (Liu et al. 2015). Briefly, commercially purchased neutravidin (Thermo Fisher Scientific) coated agarose beads (30–165 μm diameter) were used as solid-phase support. First, the neutravidin beads were washed with water, then 3 times with buffer A (10 mM Tris-HCl, 50 mM KCl, pH 8.0). The PCR product (10 ml) was incubated with neutravidin beads at room temperature overnight to immobilize the DNA template. The next day, the beads were added to a Pierce Centrifuge column (~ 30 μm pore size) and centrifuged at 500 rpm, 4 °C for 1 min. The beads were then washed 3 times with buffer A. Approximately 80% of the template could be attached to the neutravidin beads. The bead-attached templates were stable for weeks and reusable for multiple RNA preparations. In vitro transcription was used to prepare all RNA samples, which includes the following steps for a 10 ml transcription reaction. DNA templates attached neutravidin agarose beads were incubated with 80 mM HEPES-KOH (pH 7.5), 28 mM MgCl₂, 2 mM Spermidine, 40 mM DTT, 6 mM rNTPs, T7, SUPERase In RNase Inhibitor (Invitrogen) and deionized H₂O to a final volume of 10 ml for 3 to 5 h. The RNA product was purified by urea-denaturing polyacrylamide gel electrophoresis, eluted from gel by RNA elution buffer (0.3 M sodium acetate, 2 mM EDTA, pH 5.3) at 4 °C overnight and finally buffer changed to NMR buffer (10 mM potassium phosphate, 30 mM KCl, 2 mM MgCl₂, pH 6.8).