2-Aminopurine: A Probe of Structural Dynamics and Charge Transfer

Published on Web 10/12/2002

2-Aminopurine: A Probe of Structural Dynamics and Charge Transfer in DNA and DNA:RNA Hybrids Melanie A. O’Neill and Jacqueline K. Barton* Contribution from the DiVision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125 Received June 10, 2002

Abstract: Spectroscopic techniques are employed to probe relationships between structural dynamics and charge transfer (CT) efficiency in DNA duplexes and DNA:RNA hybrids containing photoexcited 2-aminopurine (Ap*). To better understand the variety of interactions and reactions, including CT, between Ap* and DNA, the fluorescence behavior of Ap* is investigated in a full series of redox-inactive as well as redox-active assemblies. Thus, Ap* is developed as a dual reporter of structural dynamics and basebase CT reactions in nucleic acid duplexes. CD, NMR, and thermal denaturation profiles are consistent with the family of DNA duplexes adopting a distinct conformation versus the DNA:RNA hybrids. Fluorescence measurements establish that the d(A)-r(U) tract of the DNA:RNA hybrid exhibits enhanced structural flexibility relative to that of the d(A)-d(T) tract of the DNA duplexes. The yield of CT from either G or 7-deazaguanine (Z) to Ap* in the assemblies was determined by comparing Ap* emission in redox-active G- or Z-containing duplexes to otherwise identical duplexes in which the G or Z is replaced by inosine (I), the redox-inactive nucleoside analogue. Investigations of CT not only demonstrate efficient intrastrand basebase CT in the DNA:RNA hybrids but also reveal a distance dependence of CT yield that is more shallow through the d(A)-r(U) bridge of the A-form DNA:RNA hybrids than through the d(A)-d(T) bridge of the B-form DNA duplexes. The shallow distance dependence of intrastrand CT in DNA:RNA hybrids correlates with the increased conformational flexibility of bases within the hybrid duplexes. Measurements of interstrand base-base CT provide another means to distinguish between the A- and B-form helices. Significantly, in the A-form DNA:RNA hybrids, a similar distance dependence is obtained for inter- and intrastrand reactions, while, in B-DNA, a more shallow distance dependence is evident with interstrand CT reactions. These observations are consistent with evaluations of intra- and interstrand base overlap in A- versus B-form duplexes. Overall, these data underscore the sensitivity of CT chemistry to nucleic acid structure and structural dynamics.

Introduction

The resemblance of the array of aromatic bases in the DNA double helix to conductive, one-dimensional aromatic crystals prompted the suggestion, almost 40 years ago, that the DNA π-stack might facilitate charge transfer (CT).1 Today, while there is a developing consensus regarding the existence and importance of long-range DNA CT,2-5 mechanistic descriptions of how charge migrates through DNA provide the focus of ongoing * To whom correspondence should be addressed: [email protected]. (1) (a) Eley, D. D.; Spivey, D. I. Faraday Soc. Trans. 1962, 58, 411-415. (b) Hoffman, T. A.; Ladik, J. AdV. Chem. Phys. 1964, 7, 84-158. (2) (a) Nu´n˜ez, M. E.; Barton, J. K. Curr. Opin. Chem. Biol. 2000, 4, 199206. (b) Nu´n˜ez, M. E.; Hall, D. B.; Barton, J. K. Chem. Biol. 1999, 6, 85-97. (c) Kelley, S. O.; Barton, J. K. Met. Ions Biol. Syst. 1999, 36, 211-249. (d) Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed. Engl. 1997, 36, 2715-2730. (3) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res. 2001, 34, 159-170. (4) (a) Schuster, G. B. Acc. Chem. Res. 2000, 33, 253-260. (b) Henderson, P. T.; Jones, D.; Hampikian, G.; Kan, Y. Z.; Schuster, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8353-8358. (5) (a) Giese, B. Acc. Chem. Res. 2000, 33, 631-636. (b) Giese, B.; Wessely, S.; Spormann, M.; Lindemann, U.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem., Int. Ed. 1999, 38, 996-998. (c) Bixon, M.; Giese, B.; Wessely, S.; Langenbacher, T.; Michel-Beyerle, M. E.; Jortner, J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11713-11716. 10.1021/ja0208198 CCC: $22.00 © 2002 American Chemical Society

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examining thermal or photoinduced CT reactions between electron donors and acceptors mediated by and/or directly involving DNA. To date, such investigations have focused on two general themes: (i) spectroscopic measurement of the rates and yields of CT between donors and acceptors separated by an intervening DNA bridge15-20 and (ii) evaluation of the yield of oxidative damage at guanine doublets (GG) following hole migration through DNA.21-24 Several examples of time-resolved measurements of fast (k ≈ 106-108 s-1) and ultrafast (k ≈ 1091012 s-1) DNA-mediated CT reactions involving covalently tethered metallointercalators, organic intercalators or end-caps, and DNA bases now exist. Similarly, countless investigations have demonstrated long-range oxidative damage to GG sites within DNA, including DNA-mediated oxidation over distances as far as 200 Å.2b,4b As a result, it is now generally accepted that charges can migrate rapidly and over significant distances through the DNA π-stack. 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A.; Arkin, M. R.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 2921-2925. (e) Arkin, M. R.; Stemp, E. D. A.; Pulver, S. C.; Barton, J. K. Chem. Biol. 1997, 4, 389400. (f) Hall, D. B.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 50455046. (g) Hall, D. B.; Holmlin, R. E.; Barton, J. K. Nature 1996, 382, 731-735. (22) (a) Sartor, V.; Boone, E.; Schuster, G. B. J. Phys. Chem. B 2001, 105, 11057-11059. (b) Ly, D.; Sanii, L.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121, 9400-9410. (c) Gasper, S. M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 12762-12771. (d) Ly, D.; Kan, Y. Z.; Armitage, B.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 8747-8748. (23) (a) Giese, B.; Amaudrut, J.; Ko¨hler, A. K.; Spormann, M.; Wessely, S. Nature 2001, 412, 318-320. (b) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950-12955. (c) Meggers, E.; Kusch, D.; Spichty, M.; Wille, U.; Giese, B. Angew. Chem., Int. Ed. 1998, 37, 460-462. (24) (a) Nakatani, K.; Dohno, C.; Saito, I. 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of CT processes in DNA,15b-f,16d,f,17a,18b,19c often represented by β.25 One explanation for the apparent disparity found in measurements of the CT distance dependence is that different experimental assemblies might operate within different regimes of a mechanistic continuum between one-step superexchange and multistep hopping.7a,d Irrespective of mechanism, however, DNA structural dynamics may modulate interactions between donor and acceptor and within the DNA bridge, thereby affecting the efficiency of CT. The role of structural dynamics in DNA CT has received considerably less attention than other mechanistic issues. The significance of a donor-DNA-acceptor structure, particularly base stacking within the assembly, is one of the most important themes that has emerged from studies of DNA CT conducted in our laboratories. Investigations of oxidative damage to GG sites have definitively established the occurrence of long-range DNA-mediated CT and have consistently demonstrated the remarkable sensitivity of these reactions to the integrity of the π-stack.21 Experiments in which base stacking interactions are altered by, for instance, mismatches,21a,26 sequence variation,21b structural perturbations21f or protein binding,27 emphasize how subtle changes in base stacking dramatically affect CT through DNA. Recent studies of CT in nonB-form DNA structures such as single strands,16e,19d,28a triplexes,28b,c crossover junctions,28d DNA:RNA hybrids28e,f and Z-DNA28g reveal also the sensitivity of CT to structural variations. Likewise, direct spectroscopic observation of CT reactions in DNA confirms that rapid, long-range CT is possible and that a well-coupled π-stack involving the redox participants and the DNA bases is indeed requisite to DNA-mediated CT.15 While a description of long-range DNA-mediated CT must incorporate strong stacking interactions, it is equally important to emphasize that these stacking interactions are not static but are modulated by structural dynamics. Time-resolved investigations of ethidium-modified DNA duplexes established that dynamic variations in stacking are crucial to DNA CT processes and suggest that base motions may serve to gate CT.15d Photoexcited 2-aminopurine (Ap*) can serve both as a sensitive probe of DNA structural dynamics and as a participant in CT reactions with DNA bases. The utility of Ap* as a probe of the DNA environment is illustrated in the diverse array of studies, including investigations of abasic sites, protein/DNA interactions, and the inclusion of nonnatural base surrogates within DNA.29 Investigations of DNA-mediated CT benefit from the ability of Ap* to probe base-base CT in structurally welldefined DNA assemblies unperturbed by auxiliary redox reagents. Thus, while the vast majority of spectroscopic and biochemical investigations of DNA-mediated CT employ pen(25) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265-322. (26) (a) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (b) Kelley, S. O.; Boon, E. M.; Barton, J. K.; Jackson, N. M.; Hill, M. G. Nucleic Acids Res. 1999, 27, 4830-4837. (c) Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew. Chem., Int. Ed. 1999, 38, 941-945. (27) (a) Rajski, S. R.; Kumar, S.; Roberts, R. J.; Barton, J. K. J. Am. Chem. Soc. 1999, 121, 5615-5616. (b) Rajski, S. R.; Barton, J. K. Biochemistry 2001, 40, 5556-5564. (28) (a) Kan, Y. Z.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121, 1085710864. (b) Nu´n˜ez, M. E.; Noyes, K. T.; Gianolio, D. A.; McLaughlin, L. W.; Barton, J. K. Biochemistry 2000, 39, 6190-6199. (c) Kan, Y. Z.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121, 11607-11614. (d) Odom, D. T.; Dill, E. A.; Barton, J. K. Nucleic Acids Res. 2001, 29, 2026-2033. (e) Odom, D. T.; Barton, J. K. Biochemistry 2001, 40, 8727-8737. (f) Sartor, V.; Henderson, P. T.; Schuster, G. B. J. Am. Chem. Soc. 1999, 121, 11027-11033. (g) Abdou, I. M.; Sartor, V.; Cao, H. C.; Schuster, G. B. J. Am. Chem. Soc. 2001, 123, 6696-6697.

Probe for DNA Structural Dynamics/Charge Transfer

dant redox reagents, which are not integral components of DNA, investigations of base-base CT in DNA involving photoexcited15b,c,30 and photoionized19 Ap have recently been initiated. Using Ap*, we have directly observed ultrafast base-base CT and evaluated the dependence of CT on distance, driving force, and intra- versus interstrand pathways.15b,c Thus, Ap* can act as a dual reporter of DNA structural dynamics and CT. Probing CT in DNA:RNA hybrids provides a means to explore the relationships between CT and nucleic acid structure.31 DNA:RNA hybrids are key intermediates in DNA synthesis, transcription, reverse transcription by retroviruses, and antisense therapy.32 Moreover, structurally, DNA:RNA hybrids are characteristically distinct from DNA duplexes. In solution, DNA:RNA hybrids exhibit a sequence-dependent range of local conformations intermediate between the A-form of RNA duplexes and the B-form of DNA duplexes.33 The global structure tends to be closer to A-form, with the RNA strand maintaining features characteristic of A-form structures (particularly the C3′-endo sugar pucker), and the DNA strand showing more structural variety. The sequence-dependent structural dynamics of DNA:RNA hybrids are also quite distinct from DNA duplexes, as revealed by base pair lifetimes34 and C2′OH dynamics and hydration.35 Studies of long-range oxidative damage at GG sites have previously revealed efficient, long-range CT through DNA:RNA hybrids.28e,f A reduction in the yield of oxidative damage in DNA:RNA hybrids relative to DNA duplexes was attributed to less efficient trapping within the A-form structure, rather than less efficacious CT.28f However, because of the nature of the experiments, it was not possible to examine charge injection and CT independently of charge trapping or to explicitly address the impact of structural dynamics. Here, we examine the fluorescence behavior of Ap-modified nucleic acid duplexes first in redox-inactive assemblies to develop a picture of the structural dynamics within DNA and DNA:RNA duplexes and then in redox-active duplexes to probe CT. Through these combined investigations, we can establish links between CT chemistry and structural dynamics. Experimental Section Materials. DNA oligonucleotides were synthesized on an ABI DNA Synthesizer using standard solid-phase techniques. Typically, syntheses (29) (a) Rachofsky, E. L.; Seibert, E.; Stivers, J. T.; Osman, R.; Ross, J. B. A. Biochemistry 2001, 40, 957-967. (b) Stivers, J. T. Nucleic Acids Res. 1998, 26, 3837-3844. (c) Holz, B.; Klimasauskas, S.; Serva, S.; Weinhold, E. Nucleic Acids Res. 1998, 26, 1076-1083. (d) Allan, W. B.; Reich, N. O. Biochemistry 1996, 35, 14757-14762. (e) Mandal, S. S.; Fidalgo da Silva, E.; Reha-Krantz, L. J. Biochemistry 2002, 41, 4399-4406. (f) Hochstrasser, R. A.; Carver, T. E.; Sowers, L. C.; Millar, D. P. Biochemistry 1994, 33, 11971-11979. (g) Singh, I.; Hecker, W.; Prasad, A. K.; Parmar, V. S.; Seitz, O. Chem. Commun. 2002, 500-501. (30) Larsen, O. F. A.; van Stokkum, I. H. M.; Gobets, B.; van Grondelle, R.; van Amerongen, H. Biophys. J. 2001, 81, 1115-1126. (31) (a) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag: New York, 1984. (b) Hartman, B.; Lavery, R.; Q. ReV. Biophys. 1996, 29, 309368. (32) (a) Adams, R. L. P.; Knowler, J. T.; Leader, D. P. The Biochemistry of Nucleic Acids, 10th ed.; Chapman and Hall: London, 1986; Chapter 6. (b) Hansen, U. M.; McClure, W. R. J. Biol. Chem. 1980, 255, 9564-9570. (c) Varmus, H. Science 1988, 240, 1427-1435. (d) Stephenson, M. L.; Zamenick, P. C. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 285-288. (33) (a) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Biochemistry 1998, 37, 73-80. (b) Gyi, J. I.; Conn, G. L.; Lane, A. N.; Brown, T. Biochemistry 1996, 35, 12538-12548. (c) Fedoroff, O. Y.; Salazar, M.; Reid, B. R. J. Mol. Biol. 1993, 233, 509-523. (34) (a) Snoussi, K.; Leroy, J.-L. Biochemistry 2001, 40, 8898-8904. (b) Maltseva, T. V.; Zarytova, V. F.; Chattopadhyaya, J. J Biochem. Biophys. Methods 1995, 30, 163-177. (35) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Nucleic Acids Res. 1998, 26, 3104-3110.

ARTICLES were conducted using 2.5 µM CPG columns (Glen Research) with automatic detritylation and cleavage from the support. Incorporation of the modified nucleosides, 2-aminopurine (Ap), inosine (I), and 7-deaza-guanine (Z) was accomplished using the respective phosphoramidites, as obtained from Glen Research. In the preparation of Z-containing oligonucleotides, a mild oxidant, (R)-10-camphorsulfonyl oxaziridine (obtained from Aldrich and used as received) was employed. Detritylated DNA oligonucleotides were twice purified on a HewlettPackard HPLC using a reverse-phase C-18 column with an acetonitrile/ 30 mM ammonium acetate gradient (0-15% acetonitrile over 35 min) and subsequently analyzed by mass spectrometry (MALDI). After purification, the oligonucleotides were resuspended in 1 mL of Millipore water, dried via speedvac, and stored at -20 °C until use. RNA oligonucleotides were obtained from Dharmacon Research in the 2′bis(acetoxyethoxy)-methyl ether (2′-ACE) form and deprotected and desalted immediately prior to use. DNA and DNA:RNA Duplexes. DNA and RNA oligonucleotides were quantified using UV-vis spectroscopy with the following extinction coefficients for the nucleotides at 260 nm:36 A ) 15 400; G ) 11 500; I ) 11 000; C ) 7400; T ) 8700; Ap ) 2500; U ) 10 210 L mol-1 cm-1. Typically, the purified oligonucleotides were dissolved in 200-700 µL of buffer (100 mM sodium phosphate, pH 7) to generate a stock solution of the order of 10-4 M. Duplex solutions were prepared by combining equimolar amounts of the desired DNA complements (typically 20-100 µL of oligonucleotide stock solution) and diluting to the requisite volume (typically 300 µL for fluorescence experiments) with buffer. Annealing was accomplished by placing the duplex solutions at 90 °C for approximately 5 min, followed by slow cooling to 4 °C over a period of 3 h using a Thermocycler. Duplex formation was evaluated by examining the temperature-dependent absorbance of Ap at 325 nm. Duplexes containing Ap-T base pairs melt cooperatively, including the Ap region which generally begins melting less than 1-3 °C below the natural bases.37 Melting temperatures obtained by monitoring at the Ap site, therefore, reflect the minimum temperatures for denaturation of the duplexes. The absorbance of Ap at 325 nm decreases as a result of the helix-to-coil transition, and duplex melting is thus characterized by hypochromicity of Ap. All duplexes displayed cooperative thermal denaturation profiles with melting temperatures (measured at 325 nm for 100 µM duplex samples) g 14 °C (Type-1 duplexes) or g 23 °C (Type-2 duplexes) and were, therefore, fully duplexed under experimental conditions (5 °C or 10 °C for Type-1 and Type-2 duplexes, respectively). The annealed duplexes were maintained at e10 °C prior to and during the experiment. Absorbance at the excitation wavelength (325 nm) was similar for each duplex ((