Guanine is the DNA base that is most susceptible to 1-electron oxidation. Once formed by photooxidation, the guanine radical has several different fates: it can either undergo back electron transfer with the photooxidant or react further to give permanent products. Here, we used the fluorescent analogue, 2-aminopurine as the photooxidant, and examined two different systems for modulating the amount of permanent products from guanine oxidation. In the first system, we monitored the formation of DNA-protein crosslinks as the trapping reaction for the guanine radical. 2-aminopurine was incorporated into a oligonucleotide duplex containing a 5’-G(X)<sub>n</sub>Z-3’ sequence (n=0 or 4, X = A or T and Z= 2-aminopurine) to determine the relative rates of back electron transfer and crosslinking to protein. Crosslinking was detected by gel shift assay. Upon 325 nm irradiation, only a small change was seen in the mobility of the n=0 duplex, whereas the n=4 (Z = T) duplex showed a decrease in bands for free DNA or protein and the appearance of lower mobility bands. This result suggests that at the longer separation distances, back electron transfer, does not compete as well with the protein trapping reaction; analogous experiments with intervening adenines are underway. In the second system, the distance between the photooxidant and the closest guanine was held constant, but the sequence was designed to either increase or decrease in guanine oxidation potential with the distance from the photooxidant, to either localize or pull away the electron hole, respectively. In this case, gel shift assays and cytochrome c oxidation assays indicated that an increased lifetime of the guanine radical, as afforded by the guanine environment, leads to more oxidative reaction with protein.

Modulating back electron transfer between guanine radicals and 2-aminopurine by variations in separation distance and local sequence in duplex DNA

251st National Meeting (2016)

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