Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

Eichhorn, Catherine D.; Feng, Jun; Suddala, Krishna C.; Walter, Nils G.; Brooks, Charles L.; Al-Hashimi, Hashim M.

Uniformly 13 C/ 15 N-labeled queC36 and C14U/C17U constructs were prepared by in vitro transcription as described previously (33) . Unlabeled wild-type (WT, 5 0 -AUAAAAAACUAA-3 0 ) and A29C (5 0 -AUAACAAA CUAA-3 0 ) RNAs were purchased from Integrated DNA Technologies (IDT) and purified using a C18 column (Waters) followed by lyophilization and reconstitution in NMR buffer (15 mM sodium phosphate, pH 6.4; 25 mM sodium chloride, 0.1 mM EDTA) containing 10% D 2 O by volume. 100% D 2 O samples were prepared by repeatedly lyophilizing the sample and replacing with 99.99% pure D 2 O (Sigma) three times. RNA concentrations ranged from 1.5 to 2.8 mM. AMP, UMP and CMP (Sigma) were directly dissolved into NMR buffer with no additional purification to 5 mM. For RDC measurements, samples were dialyzed into Millipore-purified ddH 2 O using 1 kDa dialysis tubing (Spectrum Labs), lyophilized, and reconstituted into 52.4 mg/ml Pf1 phage solution (34) (35) (36) in NMR buffer with 100% D 2 O (Asla Biotech). RNA concentrations in Pf1 phage ranged from 1.5 to 2 mM.