Selective fluorescence quenching of the 8-oxoG-clamp by 8-oxodeoxyguanosine in ODN Tamer Nasr a , Zhichun Li a , Osamu Nakagawa a,b , Yosuke Taniguchi a,b , Sayaka Ono a , Shigeki Sasaki a,b, * a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b CREST, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan article info Article history: Received 11 August 2008 Revised 3 December 2008 Accepted 6 December 2008 Available online 11 December 2008 Keywords: 2 0 -Deoxy-8-oxoguanosine Fluorescence Detection 8-OxoG-clamp Oligodeoxynucleotide abstract The 8-oxoG-clamp, a specific fluorescent probe for 8-oxo-deoxyguanosine (8-oxo-dG), was incorporated into the oligodeoxynucleotide (ODN) within or at the 3 0 -end of the purine and the pyrimidine sequences. Based on the UV-melting temperature, the 8-oxoG-clamp showed slightly lower stabilizing effects on the duplexes containing 8-oxo-dG at the complementary site than that with dG. On the other hand, 8-oxo-dG in DNA was selectively detected by fluorescence quenching of the 8-oxoG-clamp. Ó 2008 Elsevier Ltd. All rights reserved. 8-Oxo-deoxyguanosine (8-oxo-dG) is a major oxidatively dam- aged metabolite of deoxyguanosine (dG) by active oxygen species (Fig. 1), and causes G/C to T/A transversion mutations in DNA 1,2 The 8-oxo-dG level is regarded as an index of the oxidative stress of cells 3 and is believed to have relevance to some diseases 4 and aging. 5 Accordingly, analysis of 8-oxo-dG is of great significance, and a variety of methods have been developed using HPLC-EC 6 , HPLC/GC-MS, 7 antibodies, 8 etc. 9 It is also of great importance to detect 8-oxo-dG in DNA, and such a method using a sequence of reactions was reported. 10 Fluo- rescent nucleobases have been widely used for the detection and in the structural study of nucleic acids. 11 However a fluorescent probe for the detection of 8-oxo-dG in DNA is lacking. Recently, we reported the 8-oxoG-clamp as the specific fluorescent probe for 8-oxo-dG with a high discrimination ability from other nucleo- sides (Fig. 2). 12 The structural analysis by 1 H NMR has shown that the selectivity of the 8-oxoG-clamp is due to the complex formed involving multiple hydrogen bonds with 8-oxo-dG including the 7N-H of 8-oxo-dG (Fig. 2, (a)). 13 We now report that fluorescent property of the 8-oxoG-clamp and its selective quenching by 8- oxo-dG are retained in the ODN probes. The diol derivative of the 8-oxoG-clamp (1) was transformed to the corresponding 5 0 -dimethoxytrityl (DMTr)-protected 3 0 -phos- phoramidite precursor (2) by a conventional method, which was incorporated into the oligodeoxynucleotides (DNA3-5) by the DNA automated synthesizer (Scheme 1). 14 The recognition prop- erty of the 8-oxoG-clamp was first evaluated by measuring the melting temperature (T m ) using DNA 3 and 4 incorporating the 8- oxoG-clamp in the middle position of the sequence (Table 1). As already reported, 8-oxo-dG has a comparable effect on du- plex stabilization with dC and dA (T m , °C: 53, 52 or 53, 50). 15 The 8-oxoG-clamp showed a slightly lower T m value to 8-oxo-dG than to dG (T m , °C: 53 vs 51 or 51 vs 48). These results are inconsistent with our expectation based on the monomer experiments, in which the 8-oxoG-clamp showed a higher affinity to 8-oxo-dG than to dG (K s M À1 : 2.3 Â 10 6 vs 2.1 Â 10 5 ). 12 It was reported that the parent G-clamp without the N-carbobenzyloxy (CBZ) group in- creased the T m value toward dG (DT m + 18 °C) compared to the dG and 5-methyl-dC base pair. 16 Unlike the parent G-clamp, the CBZ group of the 8-oxoG-clamp might not form a hydrogen bond with 7N-H probably due to steric repulsion between the benzene ring of 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.12.036 * Corresponding author. E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki). 8-Oxo-deoxyguanosine (8-oxo-dG) Deoxyguanosine (dG) HN N N N O H 2 N HN N N H N O H 2 N O 7 8 dR dR 7 8 Figure 1. Structures of deoxyguanosine and 8-oxo-deoxyguanosine. dR: b-D-2 0 - deoxyribofuranosyl. Bioorganic & Medicinal Chemistry Letters 19 (2009) 727–730 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl