NMR spectroscopy

RNA samples used in NMR experiments were prepared by in vitro transcription as described above. ¹⁵N labeled nucleotides were purchased from Silantes. RNA folding was achieved by thermal denaturation of the RNA at a concentration of ~0.2 mM followed by tenfold dilution with ice cold water and incubation for 1 h on ice. RNA samples were exchanged with NMR buffer (25 mM Tris-HCl, pH 7.4) using centrifuge concentrator devices (Vivaspin 3000 MWCO). RNA concentration in the final NMR samples was ~0.5 mM. The homogeneity of the folding was checked via 15% polyacrylamide native gels (acrylamide:bisacrylamide = 29:1, TBE as running buffer) at room temperature and at a power ≤1 W to avoid overheating. All the samples contained 3-(trimethylsilyl)-1-propanesulfonic acid (DSS) as internal NMR reference and were dissolved in 10% D₂O/90% H₂O. KCl (50 mM final concentration) and 1 equivalent of DMHBI⁺ (ligand stock solution in d₆-DMSO, final concentration of d₆-DMSO ≤2% v/v) were added directly into the NMR tube to the folded RNA.

All the NMR experiments were performed on a Bruker Avance III 600 NMR spectrometer equipped with a DCH ¹³C/¹H cryoprobe or on a Bruker Avance III 600 NMR spectrometer equipped with a BBFO room temperature probe. The HNN-COSY experiment was performed by Dr. Helena Kovacs at Bruker Biospin (Fällanden, Switzerland) on a Bruker 700 MHz spectrometer equipped with a QCI-P cryoprobe. The spectra were acquired and processed with the software Topspin 3.2 (Bruker BioSpin, Germany). Spectra analysis was performed with the software Sparky 3.114 (Goddard, T. D.; Kneller, D. G. University of California, San Francisco). The ¹H,¹⁵N-BEST-TROSY experiments were recorded using a pulse program containing the modifications proposed by Brutscher et al^60,61. and an inter-scan delay of 0.3 s. The HNN-COSY experiment⁶² was recorded using a soft WaterGATE water suppression scheme⁶³, an HN transfer delay of 2.5 ms, and a NN transfer delay of 15 ms.