Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA

Sequence-specific labeling of DNA is of immense interest for analytical and functional studies of DNA. We present a novel approach for sequence-specific labeling of DNA using a newly designed fluorescent cofactor for the DNA methyltransferase from Thermus aquaticus (M.TaqI). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded 5'-TCGA-3' DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (AdoMet) leading to methyl group transfer. The design of a new fluorescent cofactor for covalent labeling of DNA was based on three criteria: (1) Replacement of the methionine side chain of the natural cofactor AdoMet by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to DNA. (2) The adenosyl moiety is the molecular anchor for cofactor binding. (3) Attachment of a fluorophore via a flexible linker to the 8-position of the adenosyl moiety does not block cofactor binding. According to these criteria the new fluorescent cofactor 8-amino[1"-(N"-dansyl)-4"-aminobutyl]-5'-(1-aziridinyl)-5'-deoxyadenosine (3) was synthesized. 3 binds about 4-fold better than the natural cofactor AdoMet to M.TaqI and is coupled with a short duplex oligodeoxynucleotide by M.TaqI. The identity of the expected modified nucleoside was verified by electrospray ionization mass spectrometry after enzymatic fragmentation of the product duplex. In addition, the new cofactor 3 was used to sequence-specifically label plasmid DNA in a M.TaqI-catalyzed reaction.