PAPER www.rsc.org/analyst | The Analyst SPR imaging for label-free multiplexed analyses of DNA N -glycosylase interactions with damaged DNA duplexes Christelle Corne,† a Jean-Bernard Fiche,† b Didier Gasparutto,* a Val´ erie Cunin, c Emmanuel Suraniti, b Arnaud Buhot, b Julia Fuchs, b Roberto Calemczuk, b Thierry Livache* b and Alain Favier a,c Received 21st January 2008, Accepted 3rd April 2008 First published as an Advance Article on the web 1st May 2008 DOI: 10.1039/b801086b Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2 ′ -deoxyguanosine and (5 ′ S)-5 ′ ,8-cyclopurine-2 ′ -deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5 ′ S)-5 ′ ,8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins. Introduction Oxidative damage of DNA bases results from an attack upon our genome by various carcinogenic agents such as ionizing radiation, UV-A radiation, redox metals and other processes over-stimulating macrophages. Oxygen radical species create a broad range of DNA damage: abasic sites, oxidized bases, DNA strand breaks, inter- or intra-strand cross-links, cross-links to proteins or lipids. 1,2 Many oxidized bases have been identified in DNA, such as 8-oxo-7,8-dihydro-2 ′ -deoxyguanosine (8-oxodG), thymidine glycol (Tg), 5 ′ ,8-cyclo-2 ′ -deoxyribosylpurines (5 ′ ,8- cyclo-dPurines) and more than 50 different abnormalities. 3,4 In order to counteract the potential mutagenic and cytotoxic effects of the lesions, living organisms have evolved DNA repair mechanisms including excision, reversion and recombination pathways. 5,6 Among all the existing repair processes, the base a Laboratoire des L´ esions des Acides Nucl´ eiques, SCIB/UMR E3 CEA-UJF, INAC, CEA-Grenoble, F-38054 Grenoble Cedex 9, France. E-mail: didier.gasparutto@cea.fr; Fax: +33-(0)4-38-78-50-90; Tel: +33-(0)4-38-78-45-58 b CREAB, SPrAM/UMR 5819 CEA-CNRS-UJF, INAC, CEA-Grenoble, F-38054 Grenoble Cedex 9, France E-mail: thierry.livache@cea.fr; Fax: +33-(0)4-38-78-52-29; Tel: +33-(0)4-38-78-52-29 c Centre d’Investigation Biologique - CHU, Universit´ e Joseph Fourier, Grenoble, France † These authors contributed equally to this work. excision repair (BER) pathway, which is conserved from bacteria to mammals, is the major mechanism devoted to correct these latter base lesions. 7–9 The main features of BER deal with the recognition and removal of an aberrant base from the DNA by an N-glycosylase such as the bacterial Fapy DNA N-glycosylase (Fpg, also called MutM) or its eukaryote homolog oxoguanine DNA N-glycosylase1 (OGG1), followed by incision of the resulting AP-site by an AP-endonuclease or an AP-lyase. Repair is completed in subsequent steps by processing of the resulting termini, filling of the gap by polymerases and intervention of a ligase to re-seal the repaired strand. 10 The presence of alterations within DNA is believed to be associated with cell death, aging and cancer. 11–13 Therefore, the correct functioning of the DNA repair machinery is crucial for the maintenance of genomic integrity and cell viability. Genetic studies have revealed direct correlations between defects in the repair of damaged bases and human disease. Thus several works have clearly shown that a decrease in base excision repair capabilities is linked to an increased risk of cancer (for a review see ref. 14). Paz-Elizur et al. have shown using an oligonucleotide (ODN) cleavage assay that low human oxoguanine DNA N-glycosylase1 (hOGG1) activity in blood peripheral mononuclear cells was associated with an increased risk of lung cancer. 15 Existing publications demonstrate the relationship between allelic variant or allelic loss of some genes belonging to the BER system and increased cancer risk. 16 In adults, many polymorphisms of DNA repair genes are linked 1036 | Analyst, 2008, 133, 1036–1045 This journal is © The Royal Society of Chemistry 2008