ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 329 (2004) 334–336 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.02.046 Notes & Tips Delaying photobleaching and recovering luminescence of a DNA molecular light switch in DNA analysis  Liansheng Ling, a Yaxin Jiang, a Chen Wang, a Xiaohong Fang, a,¤ Lijun Wan, a Zhike He, b Chunli Bai, a,¤ and Dongmin Chen a,c,¤ a Center of Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People's Republic of China b Department of Chemistry, Wuhan University, Wuhan 430047, People's Republic of China c The Rowland Institute at Harvard, Harvard University, Cambridge, MA 02142, USA Received 6 August 2003 Available online 6 May 2004 Luminescence photobleaching is a long-standing problem encountered in luminescence analysis. There has been a great deal of interest in elucidating the photo- bleaching of diVerent Xuorescent probes and in deriving methods to delay the photobleaching. In the single-mole- cule Xuorescence study of organic-dye-labeled mole- cules, -mercaptoethanol is often used alone or together with scavenging reagents to reduce photobleaching [1]. However, -mercaptoethanol is not always eVective in photobleaching suppression [2]. Despite the studies on photobleaching of diVerent Xuorescent probes, little about the luminescence recov- ery of the photobleached Xuorophores is known. Fluo- rescence recovery after photobleaching [3], which has been used extensively to measure translational mobility of Xuorophores, is based on the Xuorescence recovery resulting from passive diVusion and translational pro- cesses; thus it is not a direct measurement of the Xuores- cence recovery of the bleached Xuorophores. Recently, enzyme-dependent Xuorescence recovery of nicotine adenine dinucleotide by dehydrogenase was reported [4]. Up to now, there has been no report on the luminescence recovery of Xuorophores that intercalate within DNA. Ru(phen) 2 (dppx) 2+ (dppx, 7,8-dimethyl-dipyridophen- azine; phen, 1,10-phenanthroline) is one of the Ru (II)–ligand complexes that have exhibited unique photo- physical properties such as the “DNA light switching” eVect [5]. These “light switching” complexes have no lumi- nescence in aqueous solution due to quenching by hydro- gen bonding between water and phenazine nitrogens of the ligand. When they bind to DNA, the interaction between the ligand and the DNA duplex protects the phenazine nitrogens from water, leading to intense emission. As their luminescence intensity can be enhanced more than 10 4 by duplex DNA [5], they have been developed as promising DNA probes and used widely in DNA detection, DNA hybridization, and dynamics studies [6–8]. The intercalative binding mode between these complexes and DNA has been studied with various methods [9–12], yet the photo- bleaching property of these complexes is unknown. In this work, we have studied the photobleaching and lumines- cence recovery of Ru(phen) 2 (dppx) 2+ . We shall show for the Wrst time that 11-mercaptoundecanoic acid is a more eVective reducing reagent than -mercaptoethanol in delaying the luminescence photobleaching. Moreover, a signiWcant recovery of the luminescence of photobleached probes has been achieved using 11-mercaptoundecanoic acid. -DNA (Sigma) has been used as an ideal model DNA molecule for Xuorescence microscopy imaging as shown in the previous studies of DNA spin-stretching and DNA condensation [13,14]. Single -DNAs can be clearly observed under Xuorescence microscopy (Axio- vert 200, Zeiss) after being immobilized on the cover glass and stained with the synthesized Ru(phen) 2 - (dppx) 2+ [14,15]. However, the luminescence of the stained DNA disappeared within 20 s under continuous illumination at 450–490 nm as a result of photobleach- ing. This short imaging time greatly hinders the further study of single DNA molecules based on Xuorescence microscopy.  Supplementary data associated with this article can be found in the online version at 10.1016/j.yabio.2004.02.046. ¤ Corresponding authors. Fax: +86-10-62650024. E-mail addresses: xfang@iccas.ac.cn (X. Fang), clbai@iccas.ac.cn (C. Bai), chen@rowland.harvard.edu (D. Chen).