Sequence‐specific Methyltransferase‐Induced Labeling of DNA (SMILing DNA)

Abstract

A new concept for sequence‐specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S‐adenosyl‐L‐methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase‐catalyzed sequence‐specific coupling with DNA. Sequence‐specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…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. For M. Hha I and M. Mbo II, the 7 or 6 positions of that same adenine moiety appear to be feasible sites for the attachment of bulky modifications 10b. However, to the best of our knowledge, only the M. Taq I and M. Hha I enzymes have been shown to transfer an aziridine AdoMet analogue to DNA.…”

“…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.…”

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

“…This direct 12 modification of the chemistry of the nucleobase by the enzyme, distinguishes this method from 13 those that use enzymes to incorporate modified nucleotides as mentioned above. 14 The enzymes used in the so-called Sequence-specific Methyltransferase-Induced Labeling 15 (SMILing) method are DNA methyltransferases (MTases) 60 . The adenine-specific DNA 16…”

“…Originally this method was used to add methyl groups, but linear alkyl, alkenyl, and alkynyl 26 functionalization was developed opening up more possibilities 60,62,63 . In the meanwhile a 27 similar approach was developed for RNA 64,65 .…”

Estudo de sondas orgânicas e estratégias de marcação fluorescente de DNA: da fotoquímica básica à microscopia óptica de super-resolução

“…The latter, M.TaqI, is part of the restriction-modification system of the thermophilic bacteria Thermus aquaticus and it is an integrant of the MTases class that catalyses the transfer of the activated methyl group from AdoMet to the exocyclic amino group (N6) of adenine, within the double-stranded 5'-TCGA -3' palindromic target sequence. 43; 44 In this work, this enzyme was vastly used for sequence-specific DNA modification not only with methyl groups, but also with extended functionalised alkyl groups, 45 as described a posteriori in Chapter 3.…”