Varian inova spectrometer

Unless indicated otherwise, all NMR experiments were performed on a 800-MHz Agilent VNMRS with 5 mm ¹H{¹³C,¹⁵N} cryoprobe using established RNAPack experiments [54 (link)]. FIDs were processed in VNMRJ 4.2 and exported to MestreNova (MestreLab Research) and SPARKY [55 ] programs for analysis and peak assignment. 5′ SLtrunc was analyzed first, and this construct's chemical shifts were used to inform assignment of 5′ SL and 5′ SLGC. For 5′ SL and 5′ SLtrunc, 1D ¹H and SSNOESY spectra were acquired on samples in 10% D₂O; 1D ¹H, TNNOESY, and ¹H–¹³C HSQC spectra were acquired on samples in 99.99% D₂O. For 5′ SLGC, 1D ¹H, SSNOESY, and HNNCOSY [56 (link)] experiments were performed on the sample in 10% D₂O; 1D ¹H, TNNOESY, ¹H–¹³C HSQC, ¹H–¹⁵N HSQC, DQF-COSY, HCCH-TOCSY, 3D ¹H–¹⁵N NOESYHQSC, and 3D ¹H–¹³C NOESYHSQC experiments were performed on the sample in 99.99% D₂O. Furthermore, 3D HNCCCH [57 (link)] experiments were performed with 5′ SLGC in 99.99% D₂O on a 600-MHz Varian INOVA spectrometer (Agilent) with 5 mm ¹H{¹³C,¹⁵N} conventional probe. To measure residual dipolar coupling, ¹H–¹³C HSQC experiments were performed on a 500-MHz Bruker AVANCE with 5 mm 1H{¹³C,¹⁵N} cryoprobe with and without ¹³C decoupling and before and after the addition of 21 mg/ml Pf1 to 5′ SLGC. The chemical shifts for the 5′ SLGC construct are deposited (BMRB ID: 28090).