Preparation of RNA oligonucleotides

RNAs were prepared by in vitro transcription using DNA oligonucleotides containing a T7 RNA polymerase promoter sequence upstream of the desired template sequence (Supplemental Table S1). Specifically, in vitro transcription reactions were performed using laboratory-prepared bacteriophage T7 RNA polymerase (2 units µL⁻¹) in 80 mM HEPES (pH 7.5 at 23°C), 40 mM DTT, 24 mM MgCl₂, 2 mM spermidine, and 2 mM of each NTP. RNA was purified using denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE). RNA bands were visualized by UV shadowing, excised, and then eluted from the gel slice by the crush-soak method overnight in 10 mM Tris-HCl (pH 7.5 at 23°C), 200 mM NaCl, and 1 mM EDTA. The RNA was precipitated by adding two volumes of 100% ethanol and incubating at −20°C for 30 min, and then pelleted by centrifugation.

To generate RNAs ³²P-radiolabeled at the 5′-terminus, RNA was subjected to dephosphorylation using rAPid Alkaline Phosphatase (Roche Diagnostics), and then radiolabeled with [γ-³²P]-ATP at the 5′-terminus using T4 polynucleotide kinase. Radiolabeled RNAs were purified by denaturing 10% PAGE and recovered as described above.