Radical-induced purine lesion formation is dependent on DNA helical topology

AbstractHerein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO•) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]4 (TG4T), d[AGGG(TTAGGG)3] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)3A] (mutTel24) were exposed to HO• radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating ..

¹H high field electron-nuclear double resonance spectroscopy at 263 GHz/9.4 T.

We present and discuss the performance of 1H electron-nuclear double resonance (ENDOR) at 263 GHz/9.4 T by employing a prototype, commercial quasi optical spectrometer. Basic instrumental features of the setup are described alongside a comprehensive characterization of the new ENDOR probe head design. The performance of three different ENDOR pulse sequences (Davies, Mims and CP-ENDOR) is evaluated using the 1H BDPA radical. A key feature of 263 GHz spectroscopy - the increase in orientation selectivity in comparison with 94 GHz experiments - is discussed in detail. For this purpose, the resolution of 1H ENDOR spectra at 263 GHz is verified using a representative protein sample containing approximately 15 picomoles of a tyrosyl radical. Davies ENDOR spectra recorded at 5 K reveal previously obscured spectral features, which are interpreted by spectral simulations aided by DFT calculations. Our analysis shows that seven internal proton couplings are detectable for this specific radical if sufficient orientation selectivity is achieved. The results prove the fidelity of 263 GHz experiments in reporting orientation-selected 1H ENDOR spectra and demonstrate that new significant information can be uncovered in complex molecular systems, owing to the enhanced resolution combined with high absolute sensitivity and no compromise in acquisition time