Fluorescent molecular rotors detect O⁶-methylguanine dynamics and repair in duplex DNA

Alkylation at the O⁶ position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O⁶-alkylguanines by the "suicide" enzyme O⁶-methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans-stilbene (^tsT) is quenched by O⁶-methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as ∼9 Å of separation. Increased fluorescence of ^tsT or the cytosine analogue ^tsC resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.