Avance 3 hd ascend 600 mhz spectrometer

General methods were as previously described.^{29 (link)} Growth media and conditions used for E. coli and Streptomyces strains and standard methods for handling strains were those described previously, unless otherwise noted.³⁰ All DNA manipulations performed following standard procedures.^30b DNA sequencing was carried out at the U. C. Davis Sequencing Facility, Davis, CA or by Genewiz. All proteins were handled at 4 °C, unless otherwise stated. Protein concentrations were determined according to the method of Bradford,^{31 (link)} using a Tecan Infinite M200 Microplate Reader with bovine serum albumin as the standard. Protein purity and size was estimated using both SDS-PAGE gel electrophoresis and an ATKA FPLC System. Accurate protein molecular weight was determined by ESI-MS on an Agilent 6530 Accurate-Mass Q-TOF LC/MS equipped with a Phenomenex Jupiter C4 column (50 mm × 2.00 mm, 5 μm). Substrate binding assays were carried out on the Tecan Microplate Reader. ¹H and ¹³C NMR spectra were obtained on a Bruker Avance III HD Ascend 600 MHz spectrometer. GC-MS analyses were carried out using an Agilent Technologies 5977A MSD with an Agilent Technologies 7890B GC system.

Single-stranded RNA ONs (4, 9, 10 and 11) (50 μM) and 1:1 mixture of RNA ONs and their respective complementary DNA sequences (4c, 9c, 10c and 11c) (50 μM) were heated at 90 °C for 3 min in sodium phosphate buffer (10 mM, pH 7.1) containing 500 mM NaCl and 20% D₂O. The samples were slowly brought to room temperature over a period of 2 h and kept at 4 °C for 1 h before ¹⁹F NMR spectrum was recorded. In ¹⁹F studies of IRES subdomain IIa, the modified constructs (50 μM) were assembled by heating a 1:1 mixture of either 13 and 13c or 14 and 14c in sodium cacodylate buffer (10 mM, pH 6.5) containing 20% D₂O at 75 °C for 4 min. The samples were cooled to room temperature over a period of 1 h and kept on an ice bath for 1 h. ¹⁹F NMR analysis of the assembled constructs were performed either in the presence or absence of 5 mM Mg^II. Spectra of ONs were recorded at a frequency of 564.9 MHz on a Bruker AVANCE III HD ASCEND 600 MHz spectrometer equipped with BB(F) Double Channel Probe at 298 K. The following spectral parameters were used^{43 (link)}: ¹⁹F excitation pulse: 11 μs; spectral width: 21.28 ppm; transmitter frequency offset: -121.14 ppm; acquisition time: 80 ms; relaxation delay: 1.5 s and number of scans 28000. Each spectrum was processed with an exponential window function using lb = 10 Hz.

General methods were as previously described.^{3 (link),29} Growth media and conditions used for E. coli strains and standard methods for strain manipulation were as described,²⁹ unless otherwise noted. All DNA manipulations performed following standard procedures.²⁹ DNA sequencing was carried out at the U. C. Davis Sequencing Facility, Davis, CA or by Genewiz. All proteins were handled at 4 °C unless otherwise stated. Protein concentrations were determined according to the method of Bradford,^{30 (link)} using a Tecan Infinite M200 Microplate Reader with bovine serum albumin as the standard. Protein purity and size was estimated using SDS-PAGE and visualized using Coomassie Blue stain and analyzed with a Bio-Rad ChemiDoc MP System. Accurate protein molecular weight was determined by ESI-MS on an Agilent 6530 Accurate-Mass Q-TOF LC/MS. Reductase activity assays were carried out on the Tecan Microplate Reader and kinetic assays of KR-catalyzed reductions were also performed by GC/MS. ¹H and ¹³C NMR spectra were obtained on a Bruker Avance III HD Ascend 600 MHz spectrometer. A Thermo LXQ equipped with Surveyor HPLC system and a Phenomenex Jupiter C4 column (150 mm×2 mm, 5.0 μm) was utilized for analysis of diketide-ACP compounds. LC-ESI-MS-MS analysis was carried out in positive ion mode for analysis of pantetheinate ejection fragments.