Av spectrometers

Proteins were buffer exchanged to 150 mM NaCl, 20 mM Tris pH 7.0, 2 mM TCEP using an Amicon® centrifugal filter unit prior to measurements. ¹H,¹⁵N-HSQC spectra of 50 μM ROQ alone and in presence of 0.5, 1.0 and 1.3-fold amounts of ADE, CDE or tandem ADE–CDE RNA were recorded at 293 K on a 600 MHz spectrometer (Bruker). Lyophilized RNAs were dissolved in 50 mM KCl, 25 mM sodium phosphate pH 7.0 and refolded by snap cooling. 1D proton spectra and all experiments on protein-RNA complexes were recorded at 293 K. ¹H,¹H-NOESY, ¹⁵N-SOFAST-HMQC (59 (link)) and further 1D proton experiments for assignments were recorded at 283 K to stabilize structures. RNA concentrations for 2D spectra ranged from 300 to 600 μM, while for 1D experiments concentrations as low as 20 μM were used. All experiments were recorded on Bruker AV spectrometers with 600, 700, 800, 900 and 950 MHz proton Larmor frequencies equipped with triple-resonance cryoprobes. For ¹H,¹H-NOESY spectra the mixing times were set to 150 or 250 msec depending on the size of the RNA, and between 320 and 600 indirect points were used for acquisition. ¹H,¹⁵N-SOFAST-HMQC spectra were recorded to unambiguously distinguish Uracils from Guanines. All spectra were processed using Topspin 4.0.6. (Bruker) and analyzed with NMRFAM-Sparky 1.414 (60 (link)).

NMR experiments were performed on Bruker AV-spectrometers (400, 500, and 800 MHz, Bruker, Billerica, MA, USA) equipped with TXI cryoprobes. Samples were dissolved in 0.6 mL of deuterated solvent and transferred to 5 mm NMR tubes. Chemical shifts were measured with reference to the residual proton signal of the incompletely deuterated solvent. 1D selective TOCSY and NOE and 2D ¹H-¹³C HSQC and HMBC experiments were carried out using standard pulse program of Bruker.