secondary structure based on icSHAPE of pre-let-7 and hDicer-TRBP bound pre-let-7

we resolved precusor let7 (pre-let-7) and human Dicer-TRBP complex bound precusor let7 (hDicer-TRBP-pre-let-7) secondary structure based on in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE). We found the free pre-let-7 RNA is more flexible comparing with hDicer-TRBP-pre-7 in the double strand region [10-20nt and 50- 60 nt]. The pre-let-7 RNA (5ʹ-UGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCACUGGGAGAUAACUAUACAAUCUACUGUCUUACC-3ʹ) was synthesized by Genescript Inc. In order to form the hairpin pre-let-7, we heated the RNA at 90 °C for 3 min then snap-cooled it on ice for 5 min. In order to reconstitute the hDicer-TRBP-pre-let-7 complex, we mixed ~200 µg (0.8 nmol) hDicer-TRBP complex and ~54 µg (2.3 nmol) pre-let-7 RNA in 50 µl of gel filtration buffer containing 2 mM Ca2+ and incubated the mixture on ice for 40 min. After this, the mixture was applied to a Superose™ 6 Increase 5/150 GL (GE Healthcare) column installed on an ÄKTAmicro (GE Healthcare) equilibrated in gel filtration buffer containing 2 mM Ca2+. Fractions containing the hDicer-TRBP-pre-let-7 complex were collected at a final concentration of ~0.4 mg/ml. NAI-N3 modified the free pre-let-7 and hDicer-TRBP-pre-let-7 complex for 10 min in 37°C. then the RNA are purified and building library as the icSHAPE protocol.