EPR Detection of Guanine Radicals in a DNA Duplex under Biological Conditions: Selective Base Oxidation by Ru(phen)2dppz3+ Using the Flash-Quench Technique

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.