J. Phys. Chem. B 2001, 105, 11057-11059
11057
Long-Distance Radical Cation Migration through A/T Base Pairs in DNA: An Experimental Test of Theory Valerie Sartor, Edna Boone, and Gary B. Schuster* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332 ReceiVed: April 11, 2001; In Final Form: July 25, 2001
It is widely accepted that radical cations (holes) can migrate long distances in duplex DNA by a series of relatively short-range steps (hops). The mechanism for the short-range migration is not clearly understood. At one extreme, the radical cation is localized on guanines (G) and undergoes a unistep migration to a distant G by superexchange through a bridge of intervening A/T base pairs. Alternatively, the radical cation can reside on the bases of the A/T bridge, even though this appears to be prohibited by differences in oxidation potentials measured for the isolated DNA bases. We report experiments on DNA oligonucleotides in which GG steps are separated by (A/T)n bridges (n ) 2-5) and a radical cation is introduced by irradiation of a covalently linked anthraquinone derivative. Quantitative assessment of the distance dependence of radical cation migration efficiency shows that it is incompatible with a mechanism that requires hole hopping exclusively by superexchange.
DNA’s central role in molecular biology1,2 and its possible applications in materials science3-7 have focused attention on its ability to act as a medium for the transport of charge over long distances. Extensive experimental investigations have revealed that radical cations (holes) migrate through DNA by a series of relatively short-range hops.8-12 A theoretical understanding of the hopping mechanism is of deep interest.13 We have suggested formation of charge-delocalized, distorted structures (polaron-like species) that facilitate hole migration through DNA by thermally (phonon) activated processes.14-16 The extent of charge delocalization in the polaron is determined by the base sequence, and all bases can participate. Alternatively, it has been proposed that the holes are localized on guanines (or sequences with adjacent Gn steps) and migrate to distant guanines by superexchange through bridging A/T base pairs without the charge ever actually residing on the bridge.17-23 These alternative conceptions of the charge migration mechanism give rise to different expectations of the distance dependence for radical cation migration efficiency. Increasing the number of A/T base pairs between guanines is expected to result in a very rapid reduction in hopping efficiency if it must occur exclusively by superexchange.22 In contrast, if the charge can reside and be delocalized in the (A/T)n base pairs that bridge guanines, then a much milder distance dependence is expected. We report experiments herein that support the latter interpretation. We have shown that holes are injected into duplex DNA by irradiation of an anthraquinone derivative (AQ) covalently attached to a 5′-terminus.9 They migrate through the DNA and react at GG steps with water to form products that are detected as strand breaks, predominantly at the 5′-G, following treatment of the samples with piperidine.24-26 We prepared a series of DNA duplexes (see Figure 1) in which the complementary (nonAQ) strand contains seven GG steps that are separated by a varying number of A/T base pairs and the AQ-strand contains four GG steps separated by the variable sequence. These oligomers were labeled at their 5′- and 3′-termini with 32P for the complementary and AQ-strand, respectively (see Supporting Information). The duplex DNA oligomers were irradiated in
sodium phosphate buffer solutions (pH ) 7.0) for 4 min in a Rayonet photoreactor at 350 nm (only the AQ absorbs light). The irradiated samples were worked up by treatment with piperidine, subjected to polyacrylamide gel electrophoresis, and visualized by autoradiography. Experimental results are shown in Figure 2. The amount of strand cleavage at each of the GG steps was determined by phosphorimagery. Figure 3 shows these data for the 5′-labeled samples plotted as the log of the amount of strand cleavage at GG1-7 divided by the amount of strand cleavage at GG1, which is ca. 25 Å from the AQ in all cases, versus the distance from the AQ. There is a linear relationship between ln(GGn/GG1) and the distance from the AQ. And, more importantly, the slopes of these lines, including those from the 3′-labeled cases, are statistically indistinguishable from each other with a mean value of -0.02 ( 0.007 Å-1 (see Table 1). The magnitude of this slope is related fundamentally to the ratio of rates for hopping of the hole and its trapping by water.8,14 In the present case, the hole trapping rates are expected to be constant due to the similar position of each GG step in the designed sequences. Importantly, slopes of similar magnitude to those reported here have been obtained in related experiments using disparate DNA oligomers and using different techniques to assess the efficiency of charge migration.14,27-30 From these results, it is clear that hole migration is not strongly controlled by the sequence of DNA bases, and the efficiency of hole hopping depends only weakly on the number of A/T base pairs between GG steps over the range we have examined. The possibility that hopping of charge through DNA from G to G by superexchange through A/T bridges has been considered most carefully and thoughtfully by Jortner, Bixon, and their coworkers.21,22 They calculated matrix elements for hole transport between adjacent bases in duplex DNA and predicted the effects of base sequence and distance on the rate of charge transport for standard B-form DNA without the explicit consideration of molecular motion or of dynamic disorder.11 An unexpected outcome from their work is the prediction that thymine is a superior mediator of radical cation transport relative to adenine.
10.1021/jp011354v CCC: $20.00 © 2001 American Chemical Society Published on Web 10/24/2001
11058 J. Phys. Chem. B, Vol. 105, No. 45, 2001
Letters
Figure 1. Structures of DNA conjugates.
Figure 3. Plot of ln(GGn/GG1) against the distance from the AQ to the 5′-G of the GG step calculated on the basis that the distance between base pairs in B-form DNA is 3.4 Å. The data are: DNA(T2) [; DNA(T3) 9, DNA(T4) 2, and DNA(T5) b. The lines are the least-squares fit of the data including GG1/GG1.
TABLE 1: (A/T) Bridge Length and Charge Transport: Experiment and Theory
Figure 2. Autoradiograms from the irradiation of DNA(T2-T5), as described in the text. Lanes 1, 3, 5, and 7 are dark control experiments (the DNA sample was treated identically to the experimental sample except that it was never exposed to UV light) for DNA(T2-T5), respectively. Lanes 2, 4, 6, and 8 are the experiments where the DNA has been irradiated for 4 min with 350 nm lamps and worked up by treatment with piperidine.
Their approach can be applied to the DNA sequences investigated here that have GG steps separated by (T)n sequences with n varying from 2 to 5. The calculated22 unistep superexchange hole hopping rates for the examined sequences are shown in Table 1. In contrast with the experimental findings, the calculations indicate a very significant decrease in hopping rate over this series of (A/T)n bridge lengths. The experimental results do not support the view that unistep hole migration occurs exclusively by superexchange through A/T sequences. It is remarkable that the number of A/T base pairs in the “bridge” has such a small effect on the distance dependence of charge transport. Findings in different systems reported previously29,31 show a similar diminished effect of distance for hole over long (A/T) segments. In these experiments, the magnitude of the distance dependence (slope) varies
DNA sequence
VDA (×103 eV)a
V+(G,G) (×103 eV)b
rel. rate unistepc
slope (Å-1) experimentd,e
DNA(T2) DNA(T3) DNA(T4) DNA(T5)
5.13 1.35 0.35 0.093
29 10 3.5 1.2
)1.00 0.11 0.013 0.0015
-0.017 (-0.018) -0.024 (-0.022) -0.024 (-0.013) -0.022 (ND)f
a The value of V DA (the second-order electronic coupling term for charge transport) calculated according to eq 3 in ref 22. b As defined in eq 2 of ref 22, V+(G,G) is the first-order coupling matrix element for radical cation transfer from G to G, calculated with {V+(RR)(G,G)}, the matrix element for intrastrand hole transport from G to G from Table 2 of ref 22, assigned as “contact”,33 an interbase pair distance of 3.3 Å, and β ) 0.64 Å-1.12 c These “unistep” rates are calculated according to eq 8 of ref 22 with the assumption that the Franck-Condon factors (F) are constant. The values indicate that the rate of hole transport across an (A/T)n bridge should decrease ca. 600-fold if this is the only operative mechanism. d Slope of the line ln(GGn/GG1) vs distance from the AQ. The first value comes from experiments where the complementary strand is 5′-labeled with 32P, the values in parentheses are from experiments in which the 3′-terminus of the AQstrand is labeled. e Estimated errors in the slopes is ( 0.007 Å-1. f Not determined.
somewhat, but this can be due to variation in the rates of those reactions that compete with hole transport for experiments performed under different conditions.
Letters The results reported here and those from the related experiments of others29,31 demonstrate that the radical cation can reside on the bases in the A/T bridge, despite the fact that the oxidation potential (Eox) of the adenine is somewhat greater than that for guanine in solution.32 Evidently, the Eox of isolated bases are not entirely relevant to duplex DNA where each base is bracketed by two other (except at the termini) dynamically fluctuating bases, where each base is involved in hydrogen bonds with its Watson-Crick partner, and where the bases are embedded in a polymeric anion that contains dynamically varying structured clusters of water molecules and counterions. Relevant theories of charge transport in DNA must consider these facts. In summary, our results show that transport of radical cations through duplex DNA does not occur exclusively by unistep superexchange through (A/T)n bridges where n ) 2-5. The distance dependence of the reaction efficiency, as indicated by strand cleavage at GG steps, is too shallow. These findings indicate that some radical cation charge density resides on the (A/T)n bases of the bridge. Acknowledgment. This work was supported by a grant from the National Science Foundation, for which we are grateful. Dr. Nadia Boguslavsky prepared the DNA oligonucleotides. We thank Professor Uzi Landman, School of Physics, Georgia Institute of Technology, for several helpful and insightful discussions. Supporting Information Available: Experimental data for the anthraquinone strand labeled at the 3′ termini and the complementary strand labeled at the 5′ termini. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Armitage, B. Chem. ReV. 1998, 98, 1171-1200. (2) Burrows, C. J.; Muller, J. G. Chem. ReV. 1998, 98, 1109-1154. (3) Fink, H. W.; Schonenberger, C. Nature 1999, 398, 407-410. (4) Porath, D.; Bezryadin, A.; de Vries, S.; Dekker, C. Nature 2000, 403, 635-638. (5) Tran, P.; Alavi, B.; Grunner, G. Phys. ReV. Lett. 2000, 85, 15641567.
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