Probing IRES RNA Structure by 19F NMR

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.