Syto9

An MX3005p reverse transcription polymerase chain reaction (RT-PCR) instrument (Agilent) was used to perform PaSTRy^{50 (link)} experiments with the SYTO9 (Invitrogen) dye, which can detect the presence of RNA. We set up 50-µL reactions in a thin-walled PCR plate (Agilent), containing ~1 μL of either CVA10 mature virus or empty- or A-particles and 5 mM SYTO9 in PBS (pH 7.4), and the temperature ramped from 25 to 99 °C. Fluorescence was recorded in triplicate at 1 °C intervals.

Thermofluor experiments^{47 (link)} were performed with a MX3005p RT-PCR instrument (Agilent/Stratagene). Fluorescent probe SYTO9 and SYPROred (both from Invitrogen) were used to detect the presence of single-stranded RNA and exposed hydrophobic regions of capsid proteins respectively. Multiple reaction mixtures, each with a total volume of 50 μl, containing 1.0 μg of virus particles, 5 μM SYTO9 and 3 × SYPROred, but with different a pH scale (ranging 5.5 to 8.5, 0.5 interval), were set up in thin-walled PCR plates (from Agilent). The fluorescence level was recorded in triplicate at 0.5 °C intervals from 25 to 99 °C. In addition, a similar thermal stability assay was also performed on virus-Fab or virus-Ab immune complexes with 1.0 μg of CVA6 particles pre-incubated with either antibody or Fab (with a final antibody/fab concentration of 50 μg ml⁻¹) at 37 °C for 1 h. The temperatures at which RNA was released (Tr) and at which particles melted temperature (Tm) were recorded as the minimums of the negative first derivative of the RNA exposure and protein denaturation curves, respectively.