Monitoring RV-A2 RNA Accessibility via SYTO 82

Ref. [48 (link)] was performed according to Real-Hohn et al. [49 (link)] with minor adaptations. RNA accessibility was monitored with SYTO 82 (Thermo Fisher, Waltham, MA, USA) in a Bio-Rad CFX Connect Real-Time PCR instrument (Hercules, CA, USA). Purified RV-A2 (~3.5 µg) in PBS minus PDS (control) and plus PDS at 200 µM final concentration, was incubated for 4 h at 4 °C (negligible virus breathing) or 34 °C (strong virus breathing). Unbound PDS was removed by ultrafiltration in 100 K Merck Amicon Ultra Filter units, followed by four PBS washes to eliminate the remaining unbound PDS. SYTO 82 was added to a final concentration of 5 µM, and the volumes were adjusted to 70 µL with PBS. Three 20 µL aliquots from each of these samples were dispensed into the wells of a thin-walled PCR plate, the temperature was ramped from 25–95 °C at 1.5 °C/min, and SYTO 82 light-up fluorescence was recorded. Six independent measurements were made for each condition. Data were rendered as a dot plot revealing the temperature at which the RNA becomes accessible for SYTO 82 binding [49 (link)].