• RNA, acridine orange;
  • cooperative transition;
  • luminescence;
  • flow cytometry


The products of interaction between acridine orange (AO) and natural RNA, or the synthetic RNA homopolymers are precipitates insoluble over a wide range of ionic strength. These complexes have a composition of 1 AO molecule per 1 phosphatate. The reaction is highly cooperative and the complex exhibits metachromatic luminescence. Significant differences in the luminescence spectra, related to base composition of RNA, characterize the insoluble complexes. This observation suggests that dye-base interactions take place in the AO-RNA complexes.

During titration of poly(rA) with AO and simultaneous measurement of the luminescence and light scatter a two-step formation of the particles could be detected. The cooperative binding of the ligand at D/P < 0.6 coincides with formation of small particles (molecular aggregates or micelles) and is followed at D/P > 0.6 by a cooperative agglomeration; the product of this agglomeration is particles of the size 0.35 μm and larger.

Evidence is presented that suggests that the long wavelength luminescence (∼ 650 nm) is a consequence of the solute-solid state transition of the AO-RNA interaction product rather than the classic dye-dye interactions previously visualized in the stacking model. Since these novel observations cannot be fully explained by the previously postulated molecular mechanisms of AO binding, an alternative model is advanced. Its implications in quantitative cytochemistry of nucleic acid as applied to flow cytometry are discussed.