Oxidation of Guanine at a Distance in Oligonucleotides Induced by

unimolecular oxidation of guanine by 2AP radicals at a distance can also be monitored directly in ... guanines at sites distant from the 2AP residue i...
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10924

J. Phys. Chem. B 1999, 103, 10924-10933

Oxidation of Guanine at a Distance in Oligonucleotides Induced by Two-Photon Photoionization of 2-Aminopurine Vladimir Shafirovich,* Alexander Dourandin, Weidong Huang, Natalia P. Luneva, and Nicholas E. Geacintov Chemistry Department and Radiation and Solid State Laboratory, 31 Washington Place, New York UniVersity, New York, New York 10003-5180 ReceiVed: June 17, 1999; In Final Form: September 23, 1999

Photoionization of free 2-aminopurine (2AP) in aqueous solutions, or of 2AP residues in oligonucleotides, is observed upon excitation with 308 nm XeCl excimer laser pulses (fwhm ) 12 ns, e100 mJ/cm2/pulse) and involves the consecutive absorption of two photons. The absorption of light by the normal DNA bases at this wavelength is negligible under the same conditions. The kinetics and transient absorption spectra of the hydrated electrons and of 2AP radicals resulting from the deprotonation of the 2AP radical cations formed initially, have been investigated using standard spectroscopic methods. The 2AP radicals in aqueous solutions reversibly oxidize 2′-deoxyguanosine and guanosine 5′-monophosphate but do not react with the other three DNA nucleosides or nucleotides. The efficiency of photoionization of 2AP decreases in the following order: free 2AP > 2AP incorporated into single-stranded oligonucleotide > 2AP in double-stranded oligonucleotides. Photoionization of single 2AP residues incorporated into 18-mer single- and double-stranded oligonucleotides results in the oxidation of guanine residues in GG doublets. Employing gel electrophoresis methods, strand cleavage at these GG sites and at the site of the 2AP residue is observed after treatment of the irradiated oligonucleotides with hot piperidine. Using nanosecond transient absorption techniques, it is shown that the unimolecular oxidation of guanine by 2AP radicals at a distance can also be monitored directly in singlestranded oligonucleotides containing GG-doublets. It is found that increasing the number of nucleic acid bases between the 2AP radicals and the GG-doublet from 0 to 5 results in a decrease in the rate constant of guanine radical formation by a factor greater than 103.

1. Introduction

SCHEME 1

The migration of oxidative damage in double-stranded DNA molecules is currently an active area of research.1-6 A number of studies have focused on photosensitized DNA strand cleavage as a damage end point using high-resolution polyacrylamide gel electrophoresis techniques to resolve the DNA fragments of varying sizes that were formed. The irradiated oligonucleotides were treated with hot alkali in order to achieve strand cleavage at the guanine (G) sites, which are the most easily oxidizable DNA bases. For instance, pyperidine-labile strand breakage induced by photoexcitation of covalently linked rhodium metallointercalators occurs predominantly at guanines (mostly at the 5′-G in 5′-GG-3′ doublets), and extends up to 10-12 base pairs away from the rhodium intercalation sites.2a Damage migration from the site of photoinitiation consisting of a covalently linked anthraquinone photosensitizer to guanine sites has also been reported.3c The migration of DNA damage initiated by the photolysis of site-specifically modified (+)-antibenzo[a]pyrene diol epoxide-guainine residues,4 or 4′-pivaloylmodified5 oligonucleotide duplexes are further examples of DNA oxidative chemistry at a distance. In contrast to these studies in which DNA strand cleavage is the major detectable reaction, information on the dynamics of oxidative DNA damage at a distance is limited to only a few * To whom correspondence should be addressed. Phone: (212) 998 8456. Fax: (212) 998 8421. E-mail: [email protected].

spectroscopic observations of nucleoside radicals generated by laser radiation that causes the ionization of DNA bases.1 The distributions of radical species in DNA generated by 193 nm ArF excimer laser pulses have been investigated.1 This mode of excitation induces predominantly single-photon ionization of nucleic acid bases. Within the microsecond time domain (e5 µs), guanine radicals were far more dominant than suggested by their molar exctinction coefficients and the photoionization quantum yields at 193 nm relative to those of the other three DNA bases. Here we report that the site-specific insertion of a single 2-aminopurine (2AP) residue (Scheme 1) into oligonucleotides provides an opportunity for the direct spectroscopic observation of guanine oxidation at a distance using laser flash photolysis techniques. 2-Aminopurine is a mutagenic base analog7 which can substitute for adenine in double-stranded DNA without

10.1021/jp992002f CCC: $18.00 © 1999 American Chemical Society Published on Web 11/19/1999

Oxidation of Guanine in Oligonucleotides significantly altering the stability of the duplexes.8 While the normal DNA bases (A, C, G, and T) are characterized by absorption maxima around 260 nm and negligible absorbances at wavelengths g300 nm, the absorption maximum of 2AP occurs at 305 nm.9-11 This particular feature of the absorption spectrum of 2AP opens an opportunity for the site-specific excitation of the 2AP residues with 308 nm XeCl excimer laser pulses. In this article we report that intense 308 nm laser pulses induce site-selectiVe photoionization of 2AP residues in oligonucleotides and free 2AP in aqueous solutions. The fates and reactivities of the 2AP radicals can be easily monitored by transient absorption spectroscopy methods. It is shown that 2AP radicals selectively oxidize guanines and do not react significantly with any of the other nucleic acid bases (A, C, or T). The time dependence of the oxidation of guanines by the sitespecifically positioned 2AP radicals has been studied, and these results are compared with DNA strand cleavage at the same G sites. While strand cleavage is observed on both the complementary strand and the strand containing the 2AP residues, the focus of this work was only on the latter. The oxidation of the guanines at sites distant from the 2AP residue is attributed to the long-range migration of reactive intermediates from the 2AP sites to guanine residues. 2. Experimental Section Materials. 2-Aminopurine, adenosine 5′-monophospate (AMP), cytidine 5′-monophospate (CMP), guanosine 5′-monophospate (GMP), and thymidine 5′-monophospate (TMP) were obtained from Sigma Chemical Corp. (St. Louis, MO) and were used as received. The 2AP-modified and complementary 18-mer oligonucleotides were synthesized on a Cyclone DNA synthesizer (Biosearch, Inc., San Rafael, CA) using nucleoside phosphoramidites and solid supports from Glen Research (Sterling, VA). The tritylated oligonucleotides were removed from the solid support and deprotected with concentrated ammonium hydroxide according to manufacturer’s recommendations, purified by reversed phase HPLC on a PRP-1 column (Varian Associates, Walnut Creek, CA) with a linear gradient of acetonitrile/50 mM triethylammonium acetate from 8 to 40%, and detritylated in 80% acetic acid according to standard protocols. The 2APmodified oligonucleotides and the complementary strands (10 mol % excess over the concentration of the 2AP-modified oligonucleotide) were dissolved in 20 mM phosphate buffer, pH 7, containing 100 mM NaCl. Annealing of the two strands was accomplished by heating the samples to 80 °C for about 10 min, and then allowing the samples to cool slowly back to room temperature overnight. Laser Flash Photolysis. The transient absorption spectra were recorded using a kinetic spectrometer system (7 ns response time) using a pulsed Lambda Physik EMG 160 MSC XeCl excimer laser (308 nm, fwhm ) 12 ns, e80 mJ/pulse/cm2, 10 Hz) as an excitation source, and a 75 W pulsed xenon lamp as the probe flash transmitted through a McPherson monochromator. The transient absorption signals were detected by a Hamamatsu R928 phomultiplier, and its output was digitized and recorded using a Tektronix TDS 620 oscilloscope. Typically 50-500 kinetic curves, measured at a particular wavelength, were accumulated and averaged in order to improve the signal/ noise ratio. By automatically advancing the wavelength settings of the McPherson monochromator stepwise, at 5 nm intervals, arrays of 99 kinetic response curves in the 260-750 nm wavelength range were obtained. The transient absorption spectra measured at fixed, different time intervals after the

J. Phys. Chem. B, Vol. 103, No. 49, 1999 10925 excitation, ∆t, were constructed by sampling the amplitudes of each of the 99 decay curves at the same time interval ∆t. All experiments, including data collection and analysis, were controlled by an IBM PC computer. The sample solutions were either deoxygenated with nitrogen gas or saturated with oxygen gas and were pumped through a 0.5 mL quartz flow cell with an optical path length of 1 cm aligned parallel to the direction of the probe light beam. The solution flow (2-3 mL/min) was produced by positive N2 or O2 pressure and was controlled by a computer-controlled Teflon solenoid valve (Parker Hannifin Corp., Fairfield, NJ). The excimer laser light was focused with a quartz lens (focal length g 1 m) and was passed through a 3 mm × 9 mm rectangular aperture to obtain a beam of uniform intensity (e80 mJ/pulse/ cm2) at the sample cell position. The transient absorbance of the sample solutions were probed by the Xe lamp flashes through a 3 mm aperture perpendicular to the laser beam. The optical densities of the solutions at 308 nm were generally ∼0.6/ cm, corresponding to ∼0.1 mM concentration of 2AP residues (the extinction coefficient of free 2AP, 305 ) 6020 M-1 cm-1).11a All laser flash photolysis experiments were performed at 20 °C. Steady-State Spectroscopy. Routine UV absorption spectra and UV melting profiles (at 260 nm) were measured using an HP 84453 diode array spectrometer with an HP 89090A Peltier temperature control unit (Hewlett-Packard GMBH, Waldbronn, Germany). Fluorescence emission spectra were obtained by conventional excitation at 310 nm in a Hitachi model MPF-2A fluorescence spectrometer (Perkin-Elmer Corp., Norwalk, CT) which was interfaced with an IBM PC computer for data collection and analysis. Oxidative DNA Damage Assay. The oligonucleotides were labeled at their 5′ termini by using T4 polynucleotide kinase (Pharmacia, Piscataway, NJ) and [γ-32P]ATP (New England Nuclear, Boston, MA). The labeled samples were purified by polyacrylamide gel electrophoresis and desalted on a BioGel P-6 spin column (Bio-Rad, Melvillle, NY) as described previously.4a The samples were lyophilized and redissolved in 20 mM phosphate buffer, pH 7.0 containing 100 mM NaCl. The 10 µL samples containing 32P 5′-end-labeled oligonucleotide strands in square Pyrex capillary tubes with inner dimensions of 2 mm × 2 mm (Vitrocom, Inc.) that are practically transparent at 308 nm, were irradiated with 308 nm laser light. The energy dosages were measured and adjusted using a Scientech (Boulder, CO) power meter. After the irradiations, the 10 µL samples were divided into two equal portions. The first aliquot was assayed for direct strand breaks without hot piperidine postirradiation treatment. The second aliquot of the irradiated sample was heated for 30 min at 90 °C in 100 µL of a 1 M piperidine solution. The samples were vacuum-dried and assayed by a denaturing polyacrylamide gel (20% acrylamide, 19:1 bis ratio, 7 M urea), and gel electrophoresis was performed as described previously.4a The gel was vacuum-dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY). The relative intensities of each of the bands were analyzed using a Bio-Rad 250 imaging system (Bio-Rad, Hercules, CA). 3. Results 2AP-Modified Oligonucleotides. General Aspects. A series of oligodeoxynucleotides containing GG pairs at various distances from the 2AP residues were used either in the singlestranded, or in the double-stranded form in complexes with their natural complementary strands with T opposite the 2AP residues.

10926 J. Phys. Chem. B, Vol. 103, No. 49, 1999

Shafirovich et al.

TABLE 1: Parameters Characterizing Fluorescence Quenching of 2AP in Oligonucleotides and Rate Constants of A(-H•) and G(-H•) Radicals compound

F(oligo)/F0

kA or ka (s-1)

kG or kg (s-1)

kAG or kag

kGA or kga

K

2AP 1 2 3 6

1 0.068c (0.0052)d 0.029c (0.0046)d 0.055c (0.0074)d 0.19c (0.033)d

(3.0 ( 0.3) × 104

(5.0 ( 0.5) × 103 (3.3 ( 0.3) × 103 (4.5 ( 0.5) × 103 (4.7 ( 0.5) × 103

(2.7 ( 0.3) × 107a (1.0 ( 0.1) × 105b >107b (1.1 ( 0.1) × 105b

(3.0 ( 0.3) × 108a (6.2 ( 0.6) × 104b n.d. (7.1 ( 0.7) × 104b

0.09 ( 0.02 1.6 ( 0.2c n.d. 1.7 ( 0.2e

a

(6.0 ( 0.6) × 103

Units of M-1 s-1. b Units of s-1. c Single-stranded oligonucleotides. d Double-stranded oligonucleotides. e Calculated using ka for 6.

The sequences of the 2AP-containing strands are defined below.

5′-GGACATCATC[2AP]CTACAGG-3′

(1)

5′-GGACATCAGG[2AP]CTACAGG-3′

(2)

5′-GGACGGATTC[2AP]CTACAGG-3′

(3)

5′-GGACATCATCACTACAGG-3′

(4)

5′-GGACGGATTCACTACAGG-3′

(5)

5′-CCATC[2AP]CTACC-3′

(6)

The 18-mer oligonucleotides (1, 4, and 5) contain two GG doublets, the first pair at the 5′ termini, and the second at the 3′ termini. A third GG doublet is inserted between the 2AP and the GG doublets at the 5′ termini in the 18-mer oligonucleotides 2 and 3. The number of nucleic acid bases between this GG doublet and the 2AP residue varied from 0 in 2 to 4 bases in 3. The 18-mer sequences 4 and 5 served as controls with the 2AP residues replaced by adenine residues. The 11-mer oligonucleotide 6 does contain a 2AP residue but no guanines, and thus served as another kind of control sequence. The 2AP-modified oligonucleotides form stable duplexes with their complementary strands with thymine residues in the partner strand opposite the 2AP residues. In aqueous buffer solutions, pH 7.0, containing 100 mM NaCl, these duplexes exhibit single, well-defined cooperative melting behavior during heatingcooling cycles (20-80-20 °C), with typical melting temperatures of 61 °C for 1 and 63 °C for 3. Excitation of free 2AP in aqueous solution generates the wellknown fluorescence emission spectrum with a maximum at 365 nm.9-11 However, when incorporated into oligonucleotides, the 2AP fluorescence is significantly quenched by nucleic acid bases.12-19 In our experiments, the fluorescence quenching efficiencies were characterized by the fluorescence yield ratios F(oligo)/F0, where F0 and F(oligo) are the integrated fluorescence signals of free 2AP and 2AP-modified oligonucleotides, respectively. In aqueous solutions of single-stranded 18-mer oligonucleotides, these ratios are less than 0.1 (Table 1). In double-stranded oligonucleotides, fluorescence quenching is more extensive than in single-stranded molecules and the F(oligo)/F0 values decrease to [2AP•+], the recombination of 2AP•+ and eh- cannot compete with reaction 2 which is the main pathway for decay of eh-. The reduction of 2AP by eh- also manifests itself in the transient absorbance monitored at 310 nm (Figure 2, insert B). The prompt bleaching due to the formation of 2AP•+ is followed by another, further decrease in the absorbance (Figure 2, insert B) with a rate constant close to the value of ka measured at 650 nm which is attributed to the scavenging of eh- by 2AP according to eq 2. The absorbance at 650 nm measured at a delay time ∆t ) 30 ns, attributed to eh-, increases nonlinearly with the laser power as shown in Figure 3. This nonlinear power dependence is a clear indication of a multiphoton ionization process23 with the yield of eh- being proportional to I n, where I (in units of

photon s-1 cm-2) is the fluence rate. A value of n ) 1.75 ( 0.1 is estimated from the power curve (Figure 3), which is typical of a two-photon ionization process.23 The laser pulse excitation of the 2AP-containing 18-mer oligonucletides (1-3) also results in the formation of hydrated electrons, but with a lower eh- yield than in the case of free 2AP in solution under identical experimental conditions (same absorbance of 2AP at 308 nm, and the same laser fluence rate and fluence). In oder to compare the photoionization efficiencies more quantitatively, we measured the eh- yield ratios, A650(oligo)/ A650(free), where A650(free) and A650(oligo) are the absorbances of eh- measured at 650 nm and delay time ∆t ) 30 ns, in solutions of free 2AP and 2AP-containing oligonucleotides, respectively. In 2AP-containing single-stranded oligonucleotides, the A650(oligo)/A650(free) values are ∼ 0.1. In double-stranded oligonucleotides, the photoionization efficiency is even lower than in single-stranded oligonucleotides, and the A650(oligo)/ A650(free) ratio drops to ∼0.02. 2-Aminopurine Radicals in Neutral Aqueous Solutions. The primary products of 2AP photoionization are 2AP radical cations and hydrated electrons. If the 2AP•+ radical cation is to be used as an oxidant of the potential substrates (e.g., guanines), it is important that the hydrated electrons be scavenged rapidly in order to avoid their reaction with the substrates, as well as with 2AP (reaction 2). Molecular oxygen, O2, is a very efficient scavenger of hydrated electrons.22,23 The O2•- radical anion is relatively unreactive, and does not react with nucleic acid bases, at least on millisecond time scales.22a,b In our experiments on the photoionization of 2AP in aqueous solutions, we observed that, upon saturating the solutions with O2, the decay kinetics of eh- are greatly accelerated while the transient absorption spectra of the 2AP radicals (e.g., at 280, 310, 385, and 500 nm) are not affected (data not shown). In O2-saturated solutions, the rapid decay of eh- by reaction with molecular oxygen, prevents the reduction of 2AP with the formation of 2AP•- and 2APH• according to reaction 2. Thus, saturating the aqueous solutions with O2, represents an efficient method for reducing the efficiencies of these side reactions. The purine radical cations are strong Bro¨nstead acids and thus rapidly deprotonate in neutral aqueous solutions.22a,b,24 For instance, the adenosine radicals generated by reaction with SO4•radicals were identified as neutral radicals, A(-H)•. A change in pH from 1 to 13 does not affect the transient absorption spectra of these species; on the basis of this result, it was inferred that the pKa of A•+ is e 1.24 Thus, in neutral aqueous solutions, the radical cation of 2AP should also be transformed rapidly to a neutral radical,25 2AP(-H)•:

2AP•+ h 2AP(-H)• + H+

(3)

In neutral aqueous solutions, the lifetime of 2AP•+ radical cations estimated from typical deprotonation rate constants of purine radical cations25 is ∼30 ns. Therefore, we conclude that the transient absorption spectra in the ∼380-500 nm range observed on microsecond time scales (Figure 2) are mostly due to 2AP(-H)• radicals rather than to 2AP•+ radical cations. The molar extinction coefficient of 2AP(-H)• radicals at 385 nm is 385(2AP(-H)•) ≈ 8400 M-1 cm-1, a value typical of neutral purine radicals.22a,b This value of 385(2AP(-H)•) was calculated from the ratio of optical densities at 385 nm and at 310 nm (see, above). Note that the molar extinction coefficient26 of O2•- at λ > 300 nm is less than 200 M-1 cm-1; thus, corrections of absorbances due to O2•- should be less than 10%. 2-Aminopurine Radicals Oxidize Guanines. In oxygensaturated neutral aqueous solutions, 2-aminopurine radicals

Oxidation of Guanine in Oligonucleotides

J. Phys. Chem. B, Vol. 103, No. 49, 1999 10929

Figure 4. Transient absorption spectra of 2AP (0.1 mM) and guanosine 5′ monophosphate (5 mM) in oxygenated 20 mM phosphate buffer (pH 7) solutions measured with different delay times after the 308 nm XeCl excimer laser pulse excitation (70 mJ/pulse/cm2).

oxidize guanosine 5′-monophosphate, GMP. Similar results were obtained with 2′-deoxyguanosine as the electron donor. On the other hand, no reactions were observed with other nucleotides such as AMP, CMP, or TMP, or their 2′-deoxynucleosides. In this work, we therefore focused our attention on the reactions of 2AP radicals with GMP in solution and with guanine residues in oligonucleotides. The transient absorption spectra of 2AP recorded in the presence of 5 mM GMP in deoxygenated solution are shown in Figure 4. At a delay time of ∆t ) 0.5 µs, the transient absorption spectrum is very close to that of 2AP radicals observed in the absence of GMP (see, Figure 2), except that the hydrated electron spectrum >500 nm is suppressed by the scavenging of the electrons by O2. In the absence of GMP, only small changes in the absorbance at 310 nm are observed for delay times ∆t up to ∼7.9 µs (Figure 2). However, in the presence of GMP there is a rapid rise in the absorbance at this wavelength, which is accompanied by a decay of the absorbance in the 335-500 nm region of the spectrum27 (Figure 4). At the delay time ∆t ) 21 µs, a pronounced absorption band with a maximum at 310 nm is evident; this spectrum is characteristic of both the guanine radical cation, G•+, and the neutral guanine radical, G(-H)•.22a,b Since, in neutral aqueous solutions, deprotonation of G•+ occurs with a rate constant k ≈ 2 × 106 s-1,28 the guanine radicals observed in the microsecond time scale transient absorption spectra (Figure 4) are mostly neutral radicals, G(-H)•. The kinetics of the absorbance changes at 310 nm, attributed to the formation of G(-H)•, in the absence of GMP (panel A), and in the presence of a 1 mM and a 5 mM GMP concentration (panels B and C, respectively), are compared in Figure 5. When the GMP concentration is increased from 1 to 5 mM, the 310 nm absorbance rises more rapidly and the transient absorbance change reaches a higher positive, maximum value, A310(tm), after a time interval tM ≈ 20 µs. The dependence of A310(tM) on the GMP concentration is depicted in the insert in Figure 5C. The extinction coefficient of G(-H)• radicals calculated using 385[2AP(-H)•] and the A310(tM)max and A310(t)0) values, is equal to ∼6500 M-1 cm-1, and is very close to the value reported in refs 22a,b. This suggests that 2AP(-H)• radicals are almost quantitatively transformed into GMP(-H)• radicals at high GMP concentrations (g5 mM). Thus, the oxidation of guanines by 2AP(-H)• radicals can be easily monitored by transient absorption spectroscopy at 310 nm. The kinetics of the absorbance changes at 310 nm are not affected when either AMP, CMP, or TMP (up to 100 mM) are added to the 2AP solutions; furthermore, the decay rate of the

Figure 5. Kinetics of the transient absorption at 310 nm induced by 308 nm XeCl excimer laser pulse excitation (70 mJ/pulse/cm2) of 2AP (0.1 mM) and guanosine 5′ monophosphate in oxygenated, 20 mM phosphate buffer solution (pH 7): panel A, [GMP] ) 0; panel B, [GMP] ) 1 mM; panel C, [GMP] ) 5 mM. The solid lines are fits of sums of two exponential terms (eq 4) to the experimental data points. The details of the fitting procedures, as well as the values of the associated fitting parameters, are described in more detail in the Supporting Information. The insert shows the effect of the GMP concentration on the maximal optical density, A310(tM), measured at 310 nm.

2AP(-H)•, monitored in the 380-500 nm wavelength range, is not affected by the presence of nucleotides other than GMP. The observed kinetics of the transient absorbance profiles can be approximated by a sum of two exponentials,

A310(t) ) A exp (-k1t) + B exp (-k2t)

(4)

where k1 and k2 are associated with the appearance of the G(H)• signal and the decay of 2AP(-H)• due to the recovery of 2AP. The advantage of monitoring changes in absorbance at 310 nm is that both reactions, the reduction of 2AP(-H)• radicals and the formation of G(-H)• radicals, can be monitored at this wavelength. Site-Selective Oxidation of Guanines in Single-Stranded Oligonucleotides. The changes in the absorbance at 310 nm induced by 308 nm laser pulse excitation of single-stranded oligonuclotides in aqueous solutions can be used to monitor the oxidation of guanine residues by 2AP(-H)• radicals. Typical results with several different oligonucleotides are summarized in Figure 6. Laser excitation (308 nm) of the 11-mer oligonucleotide 6 containing a single 2AP residue, but no guanines, induces a prompt bleaching within the 2AP absorption band; a very slow decay of this bleaching signal on the microsecond time scale (Figure 6A) is attributed to the decay of the 2AP(-H)• radical (Figure 6A). These decay kinetics are even slower than those observed in the case of free 2AP (Figure

10930 J. Phys. Chem. B, Vol. 103, No. 49, 1999

Shafirovich et al. GG tandem bases (Figure 6) are analogous to those observed in solutions containing both 2AP and GMP (Figure 5). In double-stranded oligonucleotides, the transient absorption signals were quite low due to the low efficiency of photoionization of 2AP induced by the 308 nm nanosecond excimer laser pulses. However, as the photochemical strand cleavage experiments suggest, oxidation of guanine residues at a distance by the 2AP•+ or 2AP(-H)• radicals must be occurring since strand cleavage is induced in both single- and double-stranded oligonucleotides, though with different efficiencies (Figure 1). 4. Discussion

Figure 6. Kinetics of the transient absorbance at 310 nm induced by 308 nm XeCl excimer laser pulse excitation (70 mJ/pulse/cm2) of singlestranded oligonucleotides in oxygenated, 20 mM phosphate buffer solutions (pH 7): panel A, 11-mer oligonucleotide 6 (oligonucleotide containing 2AP, but no guanines); panel B, 18-mer oligonucleotide 3, containing three GG pairs, the closest one being separated from 2AP by the four base sequence CATC; panel C, 18-mer oligonucleotide 2 with three GG pairs, the closest one flanking 2AP on its 5′-side; panel D, 18-mer oligonucleotide 5 containing three GG pairs but no 2AP residue. The solid lines are the best fits of the sum of two exponential terms (eq 4) to the experimental data points.

5A); thus, the 2AP(-H)• radical does not appear to cause the oxidation of neighboring nucleic acid bases A, T, and C in oligonucleotide 6. Upon excitation of the 2AP residues in the 18-mer oligonucleotides 1-3 which also contain GG tandem guanine residues, the transient absorption kinetics measured at 310 nm are very different from those observed in oligonucleotide 6. For example, in the case of oligonucleotide 3 which contains three GG pairs, a prompt decrease in the absorbance at 310 nm is attributed to bleaching associated with the depletion of 2AP molecules and the formation of 2AP(-H)• radicals; this prompt decrease in the absorbance is followed by a slower rise in the absorbance signal which is attributed to the formation of guanine radicals (Figure 6B). Analogous results are observed in the case of oligonucleotide 1 (data not shown). In oligonucleotide 2, a GG pair of bases flanks the 2AP residue on the 5′-side. Upon photoexcitation of 2AP, there is no initial bleaching at 310 nm as observed in the oligonucleotides 1 and 3 in which the GG pairs are separated from 2AP by 4-5 C, T, and A bases. Instead, a prompt rise in the absorbance at 310 nm is observed which is attributed to the rapid oxidation of the 2AP-flanking GG pairs to produce G(-H)• radicals (Figure 6C). In a control experiment with the 18-mer oligonucleotide 4 which contains an adenine residue instead of 2AP, no absorbance changes are observed at 310 nm upon photoexcitation with the 308 nm laser pulses (Figure 6D). Overall, the transient absorption kinetics at 310 nm observed with oligonucleotides that contain both 2AP and

Single-Photon Excitation of 2AP-Containing Oligonucleotides. The spectroscopic characteristics of the two aminopurine isomers, 6-aminopurine (adenine) and 2-aminopurine, are quite different. In the case of adenine in aqueous solutions, the lowest energy absorption maximum occurs near 260 nm and the fluorescence quantum yield is only ∼0.0003.29 In contrast, the absorption maximum of 2AP is red shifted to 305 nm and the fluorescence emission quantum yield is 0.66-0.86.10,11 The dramatic effects of the site of amino group substitution within the purine ring system are explained in terms of the different properties of the lowest electronic transitions in adenine and in 2AP. The lowest singlet excited state of adenine has nπ* character and decays mostly via internal conversion;30 on the other hand, the lowest excited state of 2AP has ππ* character and radiative transitions of the 12AP singlet excited state to the ground state are dominant. The fluorescence of 2AP is strongly quenched by nucleic acid bases.12-19 Time-correlated single-photon counting studies12-14 have shown that the interactions of 2AP with nucleic acid bases significantly decrease the 2AP fluorescence lifetimes. While the fluorescence lifetime of free 2AP in aqueous solution is 10 ns, in double-stranded DNA the lifetime is reduced to 30-50 ps. This effect has been used extensively to study the dynamics of mismatched base pairs, local melting of DNA induced by protein binding,14 stacking interactions at abasic sites in DNA,19 and the flipping of 2AP out of the double helix in complexes with methyltransferases.17 Recently, Barton et al.2h have reported that guanine is the most efficient quencher of 2AP fluorescence in DNA duplexes and that these quenching reactions occur via electron transfer from G to 12AP. However, in this work, we focus on the two-photon absorption chemistry of 2AP, specifically photoionization processes induced by intense 308 nm laser pulses and the subsequent photochemical reactions with guanine. Consecutive Two-Photon Photoionization of 2-Aminopurine. Transient absorption measurements provide direct evidence for the photoionization of 2AP by 308 nm excimer laser pulses. The nonlinear dependence of the eh- yield on the laser pulse fluence with an intensity index, n ) 1.75 ( 0.1 (Figure 3), is a good indication of the occurrence of two-photon ionization.23 Another important observation that is consistent with this conclusion is the decreased photoionization efficiency in single- and double-stranded oligonucleotides. Both of these effects can be accounted for in terms of a consecutive twophoton-induced ionization process.32 Absorption of the first photon results in the formation of the 2AP singlet excited state, and absorption of the second photon causes photoionization according to the scheme hν

hν

2AP 98 12AP 98 2AP•+ + eh-

(5)

Previous extensive studies1,22 have shown that the energy of 193 nm photons from ArF excimer lasers, E ) 6.42 eV, is

Oxidation of Guanine in Oligonucleotides

J. Phys. Chem. B, Vol. 103, No. 49, 1999 10931 SCHEME 3

SCHEME 2

sufficient to induce single-photon ionization33 of nucleic acid bases. The energy delivered by a consecutive two-photon excitation of 2AP is E ) 7.77 eV; this is the sum of the energy of the singlet excited state of 12AP (∆E00 ) 3.74 eV) and the energy of a 308 nm photon (E ) 4.03 eV). This total energy is greater than the energy of a single 193 nm photon, and it is therefore not surprising that irradiation of 2AP or 2APcontaining oligonucleotides with intense 308 nm laser pulses causes photoionization of the 2AP residues. The lifetime of 12AP in solution, being equal to 10 ns,12 favors absorption of the second photon within the same excimer laser pulse (fwhm ) 12 ns). Reduction of the 2AP fluorescence lifetimes due to quenching processes decreases the probability of absorption of the second photon, and the eh- yield is thus diminished in double-stranded DNA relative to the yield in single-stranded oligonucleotides. Oxidation of GMP by 2AP(-H)• Radicals in Solution. Analysis of Reaction Kinetics. The effect of GMP concentration on the formation of GMP(-H)• radicals is typical of a reversible reaction and can be described by Scheme 2. In this scheme, each of the two radicals can react with the other purine by electron transfer reactions with bimolecular rate constants kAG and kGA. Other, nonspecific, competitive decay channels can be specified by the pseudo-first-order rate constants kA and kG. The kinetics of these side reactions are very complex and have not been studied in detail here. On the basis of this scheme, the decay rate constants of 2AP(-H)• and GMP(-H)• are defined by

X ) kA + kAG [GMP] Y ) kG + kGA [2AP] The [GMP] and [2AP] concentrations were considered constant because the radical concentrations (