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Journal of Nucleic Acids
Volume 2010, Article ID 632041, 9 pages
Research Article

Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of -Alkylguanine-DNA Alkyltransferase Activity

1Institute for Research in Biomedicine (IRB Barcelona) IQAC-CSIC, CIBER-BBN Networking Centre on Bioengineering Biomaterials and Nanomedicine, Cluster Building, Baldiri i Reixac 10, 08028 Barcelona, Spain
2Chemical and Physical Biology CIB (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain

Received 11 June 2010; Accepted 17 July 2010

Academic Editor: Ashis Basu

Copyright © 2010 Maria Tintoré et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Human -alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by an -methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the -methyl group.