Design of a New Fluorescent Cofactor for DNA Methyltransferases and Sequence-Specific Labeling of DNA

Abstract

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.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…The process of introducing reporter groups (e.g., biotin ( 3) , Scheme 4) to a variety of DNA sequences has been termed “sequence‐specific methyltransferase‐induced labeling” (SMILing). Many different reporters, for example, fluorescent dyes have been attached to the adenine moiety of this aziridine AdoMet analogue for application in DNA labeling 10b, 11. Modelling of the AdoMet binding pocket from crystallographic data showed that steric interactions between the cofactor analogues and the enzyme are likely a key factor determining compatibility of AdoMet analogues with specific methyltransferases.…”

“…This implies that the enzyme is therefore likely to tolerate cofactor analogues that incorporate bulky modifications at this site. Successful examples of such bulky modifications to the cofactor analogue include an azide group ( 12 , Table S1 in the Supporting Information) and a range of fluorophores 11, 12. Pljevaljčić et al.…”

“…This aziridine‐based AdoMet analogue can be used for the introduction of different functional or reporter groups through modification of the adenine moiety. One such example shows the attachment of a dansyl fluorophore at the adenine 8‐position 11. The synthesis starts from 8‐bromo‐2′,3′‐ O ‐isoporpylideneadenosine, which was treated with diaminobutane to introduce a flexible linker and was converted in a few steps (substitution and deprotection) into the N‐adenosyl aziridine derivative.…”

“…Many aziridine analogues have subsequently been successfully transferred to DNA, for example, by the adenine DNA MTases M. Eco RI12b and M. Bsec I49 and cytosine DNA MTases such as M. Hha I12a and M. Sss I 14a. In these cases, functional moieties (including fluorophores)11 were placed at various locations of the aziridine cofactor. The aforementioned study by Pljevaljčić et al., which models the cofactor binding pockets of several DNA MTases together with the cofactor analogues, concludes that 4 of the 6 screened enzymes (M. Taq I, M. Rsr I, M. Dpn M, M. Pvu II) are likely to tolerate cofactors carrying bulky modifications at the 8‐position of the cofactor adenine moiety.…”

Probing Chromatin-modifying Enzymes with Chemical Tools

“…129 The work was later expanded to show that the C-6-and C-8-position of the adenosyl moiety of N-adenosylaziridine could be functionalized with multiple reporter groups such as biotin 32 130 or fluorophores 33 131,132 while still being utilized by the M.TaqI DNMT (Figure 5d). The reactions therefore allow sequence-specific methyltransferase-induced labeling of DNA (SMILing DNA).…”