Free Radicals from Irradiated Lyophilized DNA: Influence of Water of Hydration

Abstract

Lyophilized DNA equilibrated with water vapour at various relative humidities (0–95% H₂O or D₂O) was X-irradiated at 77 K and analysed for free radicals by electron paramagnetic resonance (EPR) spectroscopy in the temperature range 77–280 K. Analysis of spectra according to variation in humidity, microwave power and temperature generally yielded a doublet and a triplet spectrum at 77 K. The doublet partially converted into the 5-thymyl radical (TH^·). DNA containing deuterated thymine (dTDNA) revealed that the doublet of ‘normal’ DNA should be composed of two similar doublets, one of which should be assigned to the thymine anion, the other possibly the cytosine anion. The triplet signal was more stable and could be related to the guanine cation or its deprotonated successor. Several other patterns were detected, among them an allyl radical in highly aquated DNA (95% humidity). Other features occurred either predominantly or exclusively in DNA equilibrated above 66% relative humidity and were ascribed to an influence of the secondary structure. Among them were components possibly indicating H- or D-addition to the cytosine base, a reaction also derivable for dTDNA. Quantitative analysis of the total radical yield and the relative TH^· concentration revealed that one cause of the dominance of the latter is its thermal stability vs. other species. The total radical concentration increases with target size up to 76% relative humidity, the hydration water forming an integral part of the DNA. At 95% relative humidity OH^· radicals are formed in addition to DNA radicals, showing that ice and hydrated DNA have separated into discrete targets.