Electrochemical Analysis of Interaction Between SYBR Green I and 7-Deazapurines

Electrochemical Analysis of Interaction Between SYBR Green I and 7-deazapurines

Dudova-Vychodilova Z., Havran L., Pivonkova H., Fojta M.

Institute of Biophysics AS CR, v. v. i.

Kralovopolska 135, 612 65 Brno

The detection of nucleic acids by fluorescent dyes such as SYBR Green I (SG I) has become increasingly importantfor a variety of analytic and diagnostic applications since its introduction in the early 1990s[1]. It has been reported that the fluorescence of some fluorescent dyes is quenched by the interaction between the dyes and nucleobases[2]. Apparently, in the fluorescence quenching of reported dyes by nucleobase analogues such as 7-deazapurines in aqueous solution plays a role an intramolecular photoinduced electron transfer, which is dependent on the oxidation and reduction potential values of the nucleobases and the dye[3]. Since it is not known a priori whether the excited dye reacts as electron donor and the nucleobase as electron acceptor or vice versa, both possibilities are considered[4].

As a continuation of our previous work[2], we focused our research on the interaction between SG I and G* or A* in PCR-amplified DNA fragments. Using titration experiments we observed a competition between unmodified and G*- or A*-substituted DNA which correlated with the observed quenching effects. Further, we studied electrochemical properties of SG I using pyrolytic graphite electrode (PGE) and observed an anodic (oxidation) signal of the dye close to +1.0 V (against Ag|AgCl|3M KCl). Using this signal and signals due to electrooxidation of purine and 7-deazapurine bases[2], we detected interactions of the dye with unmodified and modified DNA at the electrode surface.

This work was supported by a Czech Science Foundation grant No. 206/12/G151.

[1] R. P. Haugland, K. D. Larison, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 1992.

[2] aH. Pivonkova, P. Horakova, M. Fojtova, M. Fojta, Anal Chem 2010, 82, 6807-6813; bL. J. P. Latimer, J. S. Lee, Journal of Biological Chemistry 1991, 266, 13849-13851.

[3] C. A. M. Seidel, A. Schulz, M. H. M. Sauer, Journal of Physical Chemistry 1996, 100, 5541-5553.

[4] H. K. Kang, E. J. Shin, S. C. Shim, Journal of Photochemistry and Photobiology B-Biology 1992, 13, 19-28.