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EPR Detection of Guanine Radicals in a DNA Duplex under Biological Conditions: Selective Base Oxidation by Ru(phen)_2dppz^(3+)Using the Flash-Quench Technique

Schiemann, Olav and Turro, Nicholas J. and Barton, Jacqueline K. (2000) EPR Detection of Guanine Radicals in a DNA Duplex under Biological Conditions: Selective Base Oxidation by Ru(phen)_2dppz^(3+)Using the Flash-Quench Technique. Journal of Physical Chemistry B, 104 (30). pp. 7214-7220. ISSN 1520-6106. http://resolver.caltech.edu/CaltechAUTHORS:20170517-070820804

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Abstract

Continuous-wave X-band EPR spectroscopy has been employed in examining the guanine radical within a DNA duplex at ambient temperature using the flash-quench technique. Guanine was selectively oxidized by DNA-bound [Ru(phen)_2dppz]^(3+) (dppz = dipyridophenazine, phen = 1,10-phenanthroline) generated in situ by photolysis in the presence of [Co(NH_3)_5Cl]^(2+) as the oxidative quencher. An EPR signal centered at g_(iso) = 2.0048 is observed in experiments with poly(dG-dC) as substrate. Comparable signals are also detected with a 13-mer oligonucleotide duplex containing only one guanine base and with calf thymus DNA, but no signal is observed with poly(dA-dT) or poly(dI-dC). These observations reflect the base selectivity of the reaction in forming the guanine radical. With ruthenium hexaammine as oxidative quencher, no signal is observed, while, with methyl viologen, a strong signal with hyperfine pattern is seen, characteristic of the reduced viologen radical and indicating that [Ru(phen)_2dppz]^(3+) was generated. The guanine radical signal, once formed upon continuous irradiation in argon-saturated aqueous buffer solution (pH 7), decays with a half-life of 30 s, but vanishes instantaneously in the dark or upon introduction of oxygen. Spin trapping experiments with N-tert-butyl-α-phenylnitrone substantiate the selectivity in generating the guanine radical; in the presence of poly(dG-dC), calf thymus DNA, the 13-mer oligonucleotide but not with poly(dA-dT) and poly(dI-dC), the detected nitroxide EPR signals are the same with g_(iso) = 2.0059, 〈a_N〉 = 15.05 G, and 〈a_H〉 = 3.11 G. Upon titration of the ruthenium intercalator into poly(dG-dC), the signal intensity increases smoothly as the [base pair]/[intercalator] ratio decreases from 100 to 25, at which point the signal intensity decreases markedly; this result may be an indication of an antiferromagnetic exchange interaction between guanine radicals. Indeed, using the flash-quench technique, EPR spectroscopy of guanine radicals within DNA now will permit the evaluation of how radicals within the DNA base stack may be coupled under biological conditions.


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http://dx.doi.org/10.1021/jp000725pDOIArticle
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ORCID:
AuthorORCID
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2000 American Chemical Society. Received 24 February 2000. Published online 6 July 2000. Published in print 1 August 2000. We are grateful to the NIH (to J.K.B.) and NSF (to N.J.T.) for their financial support of this work. O.S. also thanks the Deutsche Forschungsgemeinschaft for a postdoctoral fellowship. We are grateful also to E.D.A. Stemp for helpful discussions and S. Jockusch and M. Kleinman for technical assistance.
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Record Number:CaltechAUTHORS:20170517-070820804
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20170517-070820804
Official Citation:EPR Detection of Guanine Radicals in a DNA Duplex under Biological Conditions:  Selective Base Oxidation by Ru(phen)2dppz3+ Using the Flash-Quench Technique Olav Schiemann, Nicholas J. Turro, and Jacqueline K. Barton The Journal of Physical Chemistry B 2000 104 (30), 7214-7220 DOI: 10.1021/jp000725p
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:77519
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:17 May 2017 16:47
Last Modified:17 May 2017 16:47

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