Figure 3.

Characterization of triplex nanoswitches as suitable substrates of methyltransferase enzymes. a) Suicide reaction of an AGT enzyme with a fluorescein derivative of O⁶-BG. b) The alkyl transfer suicide reaction between the enzyme and a DNA methylated triplex hampers the covalent bond between the enzyme and the fluorescein group. c) SDS-PAGE images of reactions in which different enzymes (i.e. hMGMT, SsOGT, E. coli Ada-C, SsOGT-H⁵) after pre-incubation with the DNA nanoswitches and then reacted with the O⁶-BG fluorescent derivative. The enzymes that have reacted with the fluorescein derivative appear as green bands on the gel. The absence of any fluorescent bands indicates that the enzyme has not reacted with the fluorescent substrate. Fluorescence-labelled triplex nanoswitches appear as red bands. The asterisk shows the higher mobility of the 2-Me Triplex nanoswitch compared to the other Triplex switches (see also Figure S14). The experiments were performed by pre-incubating for 60 min the different AGT enzymes with the relevant triplex nanoswitches and then adding the O⁶-BG fluorescent derivative. Samples were then loaded on 15% acrylamide SDS-PAGE. Gels underwent Coomassie staining for the determination and correction of the protein amount loaded.