Single-Molecule Chemistry Part II: Pathway Analysis of the Oxidation of Guanine to 8-Oxo-7,8-dihydroguanosine in an Oligonucleotide Hybrid

In a short 11 nt dye-labelled oligonucleotide hybrid, we investigated the oxidation of a single guanine, G, to 8-oxo-7,8-dihydroguanosine, 8oG, a reaction proceeding via two reaction pathways. In single-molecule fluorescence traces, we were able to detect the conversion of G to 8oG and assign different reaction pathways. Reactions are initiated by a photoinduced electron transfer to the dye, creating a G radical cation, G•+, which is in equilibrium with the neutral G radical, G(−H)•. Results demonstrate that 8oG formation via water addition to G•+ proceeds within less than the time resolution of 150 ms. The reaction of G(−H)• with superoxide is more than 1000 times slower due to the formation of an intermediate with a lifetime of minutes. The overall yield of 8oG formation is low but increases considerably upon addition of dithiothreitol, thus preferring water addition over reactions depending on the presence of oxygen. Our results highlight the advantages of single-molecule real-time technology for the investigation of chemical reactions and reaction pathways. The method enables the direct observation of product formation comparable to fast kinetic methods, including flash photolysis and laser spectroscopy, but on much longer timescales. Time-resolved measurements over minutes to hours not only allow monitoring of reaction sequences that show the formation and decay of products and intermediates, but also the order of their appearance, information that is lost in classical analysis. Product yields can be obtained by counting events, in contrast to ensemble measurements that require considerable manipulation, during which intermediates may get lost.