Only intercalating ions cause the temperature sensitivity in acridine orange-stained artificial nine-plasmid system-2

This study aimed to investigate which ions influence the temperature sensitivity of acridine orange (AO)-stained nine-plasmid system-2. At 5 ℃ and 25 ℃, the nine-plasmid system-2 was electrophoresed on an undyed agarose gel, followed by post-staining with AO solutions containing different ions to detect their effects on the temperature sensitivity. Then, UV-Vis absorption and fluorescence spectra of the system combined with various ion-AO solutions were measured at 5 ℃-45 ℃, and ANOVA was used for intergroup comparisons. At 5 ℃, post-staining with the Tris-AO solution yielded 9, 4, 4, 2, and 1 bands, while post-staining with the AMPD-AO solution produced 9, 6, 4, 3, and 1 bands. Post-staining with other ion-AO solutions resulted in unchanged band numbers at 5 °C, and no temperature sensitivity was observed. Spectral measurements demonstrated that Tris-AO and AMPD-AO solutions containing the system exhibited significant fluctuations in optical signal intensity with temperature, showing temperature sensitivity. The temperature sensitivity correlates with the interference effect of specific ions on AO insertion into DNA. The organic weak base cations Tris and AMPD, which possess approximately tetrahedral symmetric structures containing amino and hydroxymethyl groups, induce temperature sensitivity, which strengthens with higher ionic strength. Other ions can only reduce the staining effect of AO.