Acridine orange interaction with DNA: Effect of ionic strength André M. Amado a , Wallance M. Pazin a , Amando S. Ito a , Vladimir A. Kuzmin b , Iouri E. Borissevitch a, ⁎ a Departamento de Física, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Vila Monte Alegre, CEP 14040-901 Ribeirão Preto, SP, Brazil b Emanuel Institute of Biochemical Physics, Russian Academy of Science, Kosygin st. 4, 117977 Moscow, Russia abstract article info Article history: Received 19 July 2016 Received in revised form 31 December 2016 Accepted 9 January 2017 Available online 24 January 2017 Background: The study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported. Methods: The study was performed using steady-state and time resolved optical absorption and florescence, fluo- rescence correlation spectroscopy and resonant light scattering techniques. Results: The presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentra- tion the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggre- gation. Conclusions: The interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO ef- ficacy in applications. General significance: This study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Acridine orange (AO, Fig. 1SM) is an organic dye widely applied in manyareas, including engineering, medicine, and biology. Though AO has been intensively investigated during decades, a permanent interest to its study exists, which is reflected inover two thousand annual publi- cations regarding AO (according to the Web of Science and Scopus). The study of acridine dyes interaction with DNA continues to be of a great interest due to the following reasons: 1) acridines are well-known aromatic DNAintercalators [1 and references therein] possessing mutagenic and carcinogenic effects and can provoke the cell death [2–6 and references therein]; 2) acridines are well-known active agents in the photodynamic thera- py of cancer [6–10], DNA being one of the principal targets of their photodynamic action; 3) DNA complexes with intercalators are applied in nanotechnology in searching for materials promising for low-dimension electronic elements, such as transistors, diodes, etc. [11–18 and references therein]. Two mechanisms of molecular binding to DNA are known: the mol- ecule binding to DNA surface (external binding) and the molecule inter- calation between planes of the DNA base pairs. In any case, external binding shall precede intercalation. Electrostatic interaction is a princi- pal reason for binding, and external conditions, such as ionic strength, should affect it. On the other hand, it was demonstrated that cations of various metals, including those of mutagenic and carcinogenic nature, induce a displacement of acridine from DNA [19,20]. Binding to biomacromolecules creates steric obstacles for the con- tact of the bound molecule with other species, for example, with quenchers of excited states and hinders the process of quenching [21, 22]. Hence, quenching of the excited states of a compound can provide information on the mechanism of its binding to DNA. In this paper, we report on the study of spectral characteristics of ac- ridine orange (AO) and quenching of its singlet (S 1 ⁎ ) and triplet (T 1 ⁎- )excited states by the stable nitroxyl radical, 2,2,6,6-tetramethyl- piperidine-N-oxyl (TEMPO), at its binding to DNA in the function of DNA concentration and in the absence and presence of NaCl to clarify the details of the AO interaction with DNA and salt effects on this inter- action. The study was performed using optical absorption, steady-state Biochimica et Biophysica Acta 1861 (2017) 900–909 ⁎ Corresponding author at: DF, FFCLRP, Universidade de São Paulo, Av Bandeirantes 3900, Vila Monte Alegre, CEP 14040-901 Ribeirão Preto, SP, Brazil. E-mail addresses: iouribor@usp.br, iourib@ffclrp.usp.br (I.E. Borissevitch). http://dx.doi.org/10.1016/j.bbagen.2017.01.023 0304-4165/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen