Luminescence of the solid complexes of acridine orange with RNA

Abstract

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 phosphate. 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 less than 0.6 coincides with formation of small particles (molecular aggregates or micelles) and is followed at D/P greater than 0.6 by a cooperative agglomeration; the product of this agglomeration is particles of the size 0.35 micrometer and larger. Evidence is presented that suggests that the long wave length luminescence (approximately 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.

Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange

“…AO is a widely used metachromatic stain for nucleic acids, when it intercalates with DNA it fluoresces green (525 nm), and when it interacts with RNA it fluoresces red (>630 nm) [12]. Such spectral shift is also observed when AO accumulates at high concentrations in acid intracellular compartments and is attributed to the formation of di- and oligomeric aggregates [13–15].…”

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

“…Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. DNA in solution undergoes denaturation when heated, and the temperature at which half remains in double-stranded conformation while the other half is single stranded is called the melting temperature (T m ).…”

“…Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure.…”

“…Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Within this condensed aggregate, AO luminesces at red wavelengths, with maximum emission at >640 nm, an excitation peak at 440 nm, and an excitation lifetime of ß20 ns (Cui et al, 2003;Kapuscinski et al, 1982Kapuscinski et al, , 1983 Darzynkiewicz et al This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain singleversus double-stranded DNA.…”

Effect of Aggregation of Acridine Orange on the Luminescent Characteristics of Its Composites with a Zinc-Containing Coordination Polymer

“…Acridine orange has a flat aromatic ring and tends to form aggregates, the luminescence of which is characterized by a significant bathochromic shift and by reduced intensity compared with the luminescence of the monomer [18]. Such a feature makes it possible to use the luminophore for selective dyeing of nucleic acids in that the luminescent characteristics of the complexes of AO with DNA and RNA differ significantly [19,20]. In particular, the complexes of the dye with DNA are characterized by a "green" luminescence (l em about 530 nm), whereas the complexes with RNA emit light of the red region (l em about 620 nm).…”