Methylene blue (MB), a dye belonging to phenothiazinum family is one of many molecules showing high affinity to nucleic acids as they interact with DNA by several mechanisms [1]. Even though MB is typically used as a photosensitizing agent and a DNA hybridization indicator, its modes of interaction with the DNA molecules are not clearly described. Various experimental studies carried out using spectroscopic and electrochemical techniques [2], and also theoretical calculations [3] have lead to the conclusion that the dye intercalates between the bases, mainly guanine, but other types of interactions such as minor groove binding close to AT sequences and screening of charges of the phosphate groups are also possible.

The interactions between DNA and MB were extensively studied electrochemically using a decreasing reduction peak of MB as useful analytical signal [4, 5]. The goal of our work was to investigate the DNA-MB interactions by monitoring the changes in the MB oxidation signal simultaneously with the MB-DNA complex signal. The home made screen-printed (SP) three electrode system consisting of a graphite working electrode, Ag/AgCl reference electrode and a graphite counter electrode, were used to carry out square wave voltammetric (SWV) experiments. Four types of DNA were used: double stranded salmon testes DNA (dsDNA) as an example of chromosomal DNA, supercoiled plasmid pUC19 (scDNA) as an exemplary circular structure of DNA, and two 8-mer oligonucleotides – 5’GCGCGCGC3’ [d(GC)₄] and 5’ATATATAT3’ [d(AT)₄] as a short DNA chains containing only one type of nucleic bases (either guanine or adenine) possible to observe by electroanalysis. After thorough cleaning of the SP electrodes in 0.25 M acetate buffer solution containing 10 mM KCl (pH 4.75), MB aliquots were successively added to the solution, and SW voltammograms were recorded after each addition. The MB concentrations have been changed from 2 nM to 10 µM.

The use of such a wide range of MB concentrations, from nano- to micromolar, to probe the interactions with various types of DNA allowed for the detection and comparison of various signals related to DNA-MB interactions. Except for the presence of a well known MB oxidation peak at ca. -0.2 V vs. Ag/AgCl, another MB oxidation peak has been detected at +0.9 V. These signals increased significantly in comparison to signals obtained in the absence of DNA, thus proving the existence of strong interactions between the DNA bases and MB. The MB oxidation peak currents depended on the type of DNA used. The peak at -0.2 V was 1.7, 2.4, 3.5, 4.3 times higher for dsDNA, scDNA, d(AT)4 and (GC)4, respectively, as compared with the MB peak in the absence of DNA. Similarly, the peak at +0.9 V was 2.2, 2.7, 3.9, 4.6 higher for dsDNA, scDNA, d(AT)4 and d(GC)4, respectively, indicating MB interactions not only with guanine, but also with adenine in these nucleic acids. Moreover, in the case of (GC)4 oligonucleotide, a new peak appeared at ca. 0 V, apparently due to the formation of a complex of MB and guanine.

References:

[1] S. Rauf, J.J. Gooding, K. Akhtar, M.A. Ghauri, M. Rahman, M.A. Anwar, A.M. Khalid, Journal of Pharmaceutical and Biomedical Analysis 2005, 37, 205.

[2]  G.S. Bang, S. Cho, B-G. Kim, Biosens. Bioelectron. 2005, 21, 863.

[3]  R. Rohs, H. Sklenar, J. Biomol. Structure and Dynamics 2004, 21, 699.

[4]  K. Kerman, D. Ozkan, P. Kara, B. Meric, J.J. Gooding, M. Ozsoz, Anal. Chim. Acta 2002, 462, 39.

[5]  A.A. Gorodetsky and J.K. Barton, Langmuir 2006, 22, 7917.

• Legal notice:
  

  Copyright (c) Pielaszek Research, all rights reserved.
  The above materials, including auxiliary resources, are subject to Publisher's copyright and the Author(s) intellectual rights. Without limiting Author(s) rights under respective Copyright Transfer Agreement, no part of the above documents may be reproduced without the express written permission of Pielaszek Research, the Publisher. Express permission from the Author(s) is required to use the above materials for academic purposes, such as lectures or scientific presentations.
  In every case, proper references including Author(s) name(s) and URL of this webpage: https://science24.com/paper/21991 must be provided.

1. Detection of supercoiled and linear plasmid DNA
2. Incorporation of RuSe_x/C within nanostructural Ir matrices to enhance oxygen reduction
3. Electrochemical screen printed graphite sensor in investigation of interactions between methylene blue and DNA
4. Miniature planar ion selective chloride electrodes and their application to blood serum analysis
5. Some electrochemical properties of laccase immobilised on the Au, IrO_x, or C₆₀-Pd polymer electrode supports
6. Screen-printed Voltammetric Genosensors for Detection of Listeria Monocytogenes in Food