Ultrafast transient-absorption and steady-state fluorescence measurements on 2-aminopurine substituted dinucleotides and 2-aminopurine substituted DNA duplexes Olaf F. A. Larsen,y a Ivo H. M. van Stokkum, a Frank L. de Weerd, a Mikas Vengris, a Charuvila T. Aravindakumar, a Rienk van Grondelle, a Nicholas E. Geacintov b and Herbert van Amerongen c a Division of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands b Department of Chemistry, New York University, New York, NY 10003 c Laboratory of Biophysics, Department of Agrotechnology and Food Sciences, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands Received 30th July 2003, Accepted 3rd November 2003 F|rst published as an Advance Article on the web 20th November 2003 Ultrafast transient-absorption and steady-state fluorescence measurements have been performed on dinucleotides comprising the fluorescent adenine analogue 2-aminopurine and guanine, adenine, cytosine, thymine or hypoxanthine, respectively. Two oligodeoxyribonucleotide duplexes that were site-selectively substituted with a single 2-aminopurine moiety were also studied. A strong quenching of the steady-state fluorescence was observed in all samples. The transient-absorption spectra were remarkably similar to those of the isolated 2-aminopurine (Larsen et al.; O. F. A. Larsen, I. H. M. van Stokkum, M.-L. Groot, J. T. M. Kennis, R. van Grondelle and H. van Amerongen, Chem. Phys. Lett., 2003, 371, 157–163), exhibiting both a fluorescent and a non-fluorescent excited state. There was no evidence for significant amounts of charge- separated states in the transient-absorption spectra. The probability that an excitation of 2AP leads to stable charge transfer products was estimated to be very low (0.1%). In the systems we studied, the observed fluorescence quenching can largely be explained by a shift of the equilibrium between the two excited states in 2AP, in which the non-fluorescent state is favored. 1. Introduction The study of charge-transfer processes in DNA is a topic of intense interest. 1–5 The fluorescent adenine analogue 2-amino- purine (2AP) has often been used to study DNA electron transfer (ET) as well as DNA structural dynamics and hetero- geneity. 1–4,6,7,18–20 The 2AP probe possesses several highly attractive properties. Contrary to the ultrashort excited-state lifetimes of the normal DNA bases, which are typically on the order of hundreds of femtoseconds (fs), 8,9 the excited-state lifetime of 2AP is relatively long, namely 12 nanoseconds (ns). In aqueous environment (pH ¼ 7) 2AP is highly fluores- cent with a quantum-yield of 0.66, and can be selectively excited because of its long wavelength absorption band around 305 nm. 10,11 Furthermore, 2AP can be incorporated into DNA, replacing a specifically chosen adenine residue for example, without significantly disturbing the B-DNA helix. 12 Strong quenching of the 2AP fluorescence upon its incor- poration into DNA has been attributed to ET processes between the normal DNA bases and the excited 2AP. 1,4,17,21 Among the normal DNA bases and based on its oxidation potential, guanine (G) is expected to be the most favorable candidate for transferring an electron to an excited 2AP resi- due. Recently, a structural heterogeneity of the excited state of 2AP has been revealed using ultrafast transient-absorption spectroscopy. 13 Besides the well-known fluorescent state of 2AP, an additional long-lived non-fluorescent state was also observed, which was populated to a significant extent after excitation. Using two-photon ionization of 2AP, ET from nearby gua- nine moieties to ionized 2AP in DNA was unambiguously demonstrated using transient-absorption techniques. 3 In this work, however, we focus on potential ET dynamics between the normal DNA bases and the single-photon excited state of 2AP. Ultrafast transient-absorption as well as steady-state fluorescence techniques have been used. Both single-stranded DNA dinucleotides comprising 2AP and guanine (G), adenine (A), thymine (T), cytosine (C), or inosine (I), respectively, were studied, as were short oligodeoxyribonucleotide duplexes that contained a single 2AP residue. A very strong quenching of the 2AP fluorescence was observed in all of these model systems. However, no evidence for a complete charge-separation due to ET was found. The transient-absorption spectra were highly similar to those of isolated 2AP and did not reveal the spectra of the expected ET products. As in isolated 2AP, in addition to the fluorescent state, a non-fluorescent electronic excited state was present. The observed fluorescence quenching was attri- buted to an equilibration between these two states, with the non-fluorescent state being present in greater proportion in the DNA system than in isolated 2AP. 2. Experimental 2.1. Chemicals 2AP DNA dinucleotides were obtained from Biolegio (Malden, The Netherlands), with a purity of 99%. The dinucleotides were dissolved in a 20 mM phosphate buffer with y Present address: Institute for Atomic and Molecular Physics (AMOLF), Kruislaan 407, 1098 SJ Amsterdam, The Netherlands; E-mail: o.larsen@amolf.nl PCCP www.rsc.org/pccp RESEARCH PAPER 154 Phys. Chem. Chem. Phys. , 2004, 6, 154–160 This journal is Q The Owner Societies 2004 DOI: 10.1039/b308992d