Updating Ortho- and Metachromatic Acridine Orange Fluorescence in Cytochemical Chromosome Staining: A Proposal for Understanding Its Differential Fluorescence on Double- and Single-Stranded Nucleic Acids Substrates Based on Intercalation

Many fluorophores display interesting features that make them useful biological labels and chemosensors, in particular in Cell Biology. Changes in the absorption-emission spectra (ortho- and metachromasia) are accounted among them. Acridine orange (AO) is one such fluorochromes that shows a prototypical orthochromatic vs. metachromatic behavior depending on its concentration and binding mode to different cellular substrates. Here, we revisit the differential AO fluorescence that occurs in selected biological examples, which allows for the identification of single-stranded or double-stranded nucleic acids. Although known for long, the ultimate reason for this phenomenon has not been properly advanced. We provide a potential molecular mechanism that adequately accounts for the different aspects of the phenomenon. This theoretical mechanism implies a difference in the degree of overlap of excited state orbitals whenever AO molecules are interacting with a single-stranded or a double-stranded nucleic acid. In the first case, massive pi-electron overlapping between bases and intercalated AO leads to a metachromatic red emission. On the contrary, no excited-state orbital overlapping in AO-intercalated DNA duplexes is possible due to excessive separation between AO molecules and compliancy to the nearest neighbor exclusion principle, which manifests as orthochromatic green fluorescence.

Automated summary

Many fluorophores, including acridine orange, exhibit orthochromatic vs. metachromatic behavior depending on their concentration and binding mode to cellular substrates. This article revisits the differential acridine orange fluorescence in selected biological examples, which can be used to identify single-stranded or double-stranded nucleic acids. The authors propose a potential molecular mechanism involving the degree of overlap of excited state orbitals.