All ε NMR samples were prepared by in vitro transcription (as described in Section 4.3 ) and were dialyzed into the NMR buffer (10 mM Na3PO4, pH 6.7, and 0.1 mM EDTA). NMR titration experiments were performed on unlabeled FL ε (Figure 1 a) and modular constructs PL ε and AL ε (Figure 5 a). Daclatasvir was dissolved in DMSO-d6 to make a 10 mM stock solution. Daclatasvir (100 µM) was screened by titrating against ε samples (50 µM), and therefore, the final NMR samples contained 1% DMSO-d6. 1H NMR experiments were used to monitor CSPs of imino protons (i.e., guanosine-H1 and uridine-H3). All NMR data were collected on an Avance III Bruker Ultrashield 600 MHz spectrometer equipped with a triple-resonance cryogenic probe. Spectra were collected at 25 °C with a recycle delay of 1.5 s and analyzed using TopSpin 4.0.
Ultrashield 600 mhz spectrometer
Manufactured by Bruker
The Ultrashield 600 MHz spectrometer is a high-performance Nuclear Magnetic Resonance (NMR) instrument manufactured by Bruker. It operates at a frequency of 600 MHz and is designed for advanced spectroscopic analysis of chemical and biological samples.
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Lab products found in correlation
2 protocols using ultrashield 600 mhz spectrometer
1
Probing ε RNA-Daclatasvir Interactions
2
Spectroscopic Analysis of Chemical Compounds
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An Agilent
Technologies spectrophotometer (type: Cary 60 UV–Vis), in MeOH;
λmax [nm] (εrel); 10 × 10 mm2 UV cells (quartz). 1H- and 13C-nuclear magnetic spectroscopy (NMR): Bruker (Ultrashield
600 MHz) spectrometer, δ in ppm with δ (1HDO)
= 5.00 ppm40 (link) at 5 °C, JHH (Hz); 13C signal assignment from heteronuclear 1H- and 13C-hetero single quantum correlation (HSQC)
and HMBC experiments. LC–MS: a Shimadzu HPLC
system, an LC-20AD pump, a DGU-20A5 online degasser unit, an SPD-M20A
diode array detector, a Rheodyne injection valve with a 200 μL
loop; column: Phenomenex Hyperclone column ODS 5 μm 250 ×
4.6 mm2 i.d., protected with a Phenomenex ODS 4 ×
3 mm2 i.d. precolumn; flow rate 0.5 mL min–1; solvent A: 4 mM aq ammonium acetate, solvent B: MeOH; solvent composition A/B (v/v): 0–5 min: 80/20; 5–55
min: 80/20–30/70; 55–60 min: 30/70–0/100; 60–70
min: 0/100; 70–75 min: 0/100–80/20. Data were collected
and processed using Shimadzu LC Solution software (version 1.24 SP1).
Technologies spectrophotometer (type: Cary 60 UV–Vis), in MeOH;
λmax [nm] (εrel); 10 × 10 mm2 UV cells (quartz). 1H- and 13C-nuclear magnetic spectroscopy (NMR): Bruker (Ultrashield
600 MHz) spectrometer, δ in ppm with δ (1HDO)
= 5.00 ppm40 (link) at 5 °C, JHH (Hz); 13C signal assignment from heteronuclear 1H- and 13C-hetero single quantum correlation (HSQC)
and HMBC experiments. LC–MS: a Shimadzu HPLC
system, an LC-20AD pump, a DGU-20A5 online degasser unit, an SPD-M20A
diode array detector, a Rheodyne injection valve with a 200 μL
loop; column: Phenomenex Hyperclone column ODS 5 μm 250 ×
4.6 mm2 i.d., protected with a Phenomenex ODS 4 ×
3 mm2 i.d. precolumn; flow rate 0.5 mL min–1; solvent A: 4 mM aq ammonium acetate, solvent B: MeOH; solvent composition A/B (v/v): 0–5 min: 80/20; 5–55
min: 80/20–30/70; 55–60 min: 30/70–0/100; 60–70
min: 0/100; 70–75 min: 0/100–80/20. Data were collected
and processed using Shimadzu LC Solution software (version 1.24 SP1).
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