All detections were conducted on a Bruker Avance III HD 850 MHz spectrometer equipped with a TCI cryoprobe (Bruker BioSpin, Germany) at 25 °C. One-dimensional 1H spectra were acquired using the standard pulse sequence NOESYGPPR1D [RD-G1-90°-t1-90°-τm-G2-90°-ACQ]. The water resonance was suppressed during the relaxation delay and mixing time, and pulsed gradients G1 and G2 were used to improve the quality of water suppression. The following experimental parameters were used: spectral width: 20 ppm, acquisition time: 2.66 s, relaxation delay: 4 s and transients: 64.
Avance 3 hd 850 mhz spectrometer
Manufactured by Bruker
Sourced in Germany
The Avance III HD 850 MHz spectrometer is a high-field nuclear magnetic resonance (NMR) instrument designed for advanced spectroscopic analysis. It features a superconducting magnet that generates a strong magnetic field of 850 MHz, enabling the acquisition of high-resolution NMR data. The spectrometer is capable of performing a variety of NMR experiments and is suitable for a wide range of applications in fields such as chemistry, materials science, and biochemistry.
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6 protocols using avance 3 hd 850 mhz spectrometer
1
NMR Spectroscopy of Biomolecules
2
NMR Spectroscopy of Uniformly Labeled Proteins
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Assignment experiments were recorded on a Bruker Avance III HD 850 MHz spectrometer using a wide bore H/X/Y/D 3.2 mm probe. MAS spinning frequency was set to 12.5 kHz, the temperature of the cooling gas was set to 310 K. The sample was u-[13C,15N]-labelled. INEPT delays were set to 2.7 ms for the H/N transfer, 10 ms for the N/C, 4.5 ms for the CO-CA transfer, and 11.2 ms for the CB/CA and CA/N simultaneous transfer. The HNCA spectrum99 was acquired in 20 h and was sampled to 18.6 ms in 15N, 24.8 ms in 13C, and 27.0 ms in the 1H dimension. The HN(CO)CA experiment99 was acquired using the same sampling as the HNCA experiment. For the HNCACB experiment99 , acquired in 32 h, sampling was identical to the previous experiments in the 1H and 15N dimensions, and 6.0 ms in the 13C dimension. Although these acquisition times in the direct and indirect dimensions are not sufficient to take full advantage of the narrow linewidths offered by the sample, they provide sufficient resolution to accomplish the sequential assignment of the repeating units.
3
Optimized Folding Protocol for G-Quadruplexes
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For the experiments described in Supplementary Figure S17 , 80 μl of a 200 μM solution of the G-quadruplex in Milli-Q water was heated at 65°C for 5 min and cooled at room temperature for 5 min. A solution containing 40 μl of 4× buffer (800 mM KCl, 4 mM MgCl2, 80 mM d-Tris, pH 7.1), 24 μl of Milli-Q water, and 16 μl of deuterated water was then added. Final concentrations were 100 μM DNA, 200 mM KCl, 1 mM MgCl2, 20 mM d-Tris, pH 7.1, and 10% D2O in a volume of 160 μl. After incubating for 30 min at room temperature, samples were heated in a thermal cycler at 97°C for 30 min, 95°C for 1 min and cooled to 25°C at a rate of 1°C/min. 1D 1H spectra were measured on a Bruker Avance III HD 850 MHz spectrometer. This folding protocol somewhat changed the NMR spectrum of A1, but significantly improved the spectra of some of the other monomeric G-quadruplexes shown in Supplementary Figure S17 .
4
Spectroscopic Characterization of Compounds
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Optical rotations were measured using a JASCO P-2000 polarimeter with a 1-cm cell. The 1H NMR spectra were measured with the Bruker Avance III HD 850 MHz spectrometer at the National Center for Inter-University Research Facilities (NCIRF) at Seoul National University; the 13C and 2D NMR spectra were recorded on the Bruker-800 MHz and JEOL JNM-ECA-600 MHz NMR spectrometers, respectively, at the College of Pharmacy, Seoul National University. Chemical shifts of all NMR spectra were referenced to the residual protonated solvent peaks for CDCl3 (δH/δC, 7.26/77.0). Low-resolution electrospray ionization (ESI) LC/MS data were acquired on an Agilent Technologies 6130 quadrupole mass spectrometer coupled with an Agilent Technologies 1200-series HPLC. High-resolution fast atom bombardment (HR-FAB) mass spectra were obtained using a JEOL JMS-600W high-resolution mass spectrometer at NCIRF. High-resolution electrospray ionization (ESI) LC/MS and ESI MS/MS data were obtained using an AB SCIEX Q-TOF 5600 high-resolution mass spectrometer at the National Instrumentation Center for Environmental Management (NICEM, Seoul, South Korea).
5
Quantifying Protein-ATP Affinity via NMR
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All data for cN-II proteins were collected at 25 °C using a Bruker Avance III™ HD 850 MHz spectrometer equipped with a 5 mm CPTCI 1H/13C/15N/D Z-GRD cryoprobe. In order to compare the relative ATP affinities towards the cN-II variants, 1H STD NMR spectra were acquired using 350-μl samples of 1.5 μM cN-II proteins dissolved in 50 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl, 2 mM TCEP, and 5 % D2O/95 % H2O. The frequency used for non-selective irradiation of the protein signals was 666 Hz (0.78 ppm), using a 50-ms shaped pulse Eburp2.1000 at a power of 40 dB. The spectra were acquired with a free induction decay (FID) resolution of 0.83 Hz, and the typical experimental time was 20 minutes.
6
High-Resolution NMR Spectroscopy Protocol
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1H NMR analyses were conducted at 298 K using a Bruker AVANCE III HD 850 MHz spectrometer (Bruker BioSpin, Germany) equipped with a TCI cryoprobe at 25 °C. One-dimensional (1D) 1H NMR spectra were acquired employing the Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence [RD-90°-(τ-180°-τ)n-ACQ] with water suppression. RD represented the relaxation delay (4 s) and τ denoted the spin echo delay (300 μs). A total of 64 transients were accumulated into 64 K data points with a spectrum width (SW) of 20 ppm and an acquisition time (ACQ) of 2.73 s.
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