University of Cambridge > Talks.cam > Keyser Group Seminar > Sequence-specific labeling of DNA with methyltransferases and synthetic cofactor analogues

Sequence-specific labeling of DNA with methyltransferases and synthetic cofactor analogues

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Sequence-specific labeling of long DNA is of major interest for functional studies of DNA and DNA -modifying enzymes as well as for DNA mapping, gene delivery and (nano)-biotechnology. We employ DNA methyltransferases (MTases) which transfer the activated methyl group of the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet or SAM ) to adenine-N6, cytosine-N4 or cytosine-C5 within specific double-stranded DNA sequences ranging from two to eight base pairs. Since the methyl group is not an attractive reporter group, we have synthesized two classes AdoMet analogues which serve as new cofactors for sequence-specific DNA functionalization and labeling by DNA M Tases. In the first class of cofactor analogues the amino acid side chain of AdoMet is replaced with an aziridinyl residue and reporter groups are attached to the adenine ring. DNA M Tase-catalyzed nucleophilic attack and aziridine ring opening leads to sequence-specific coupling of the whole aziridine cofactor with DNA and hence to covalent DNA labeling (Sequence-specific Methyltransferase-Induced Labeling of DNA , SMILing DNA ). In the second class of cofactor analogues the methyl group of AdoMet is replaced with extended carbon chains carrying an unsaturated bond in β-position to the sulfonium center (double-activated AdoMet analogues). Enzymatic transfer of extended chains containing chemical reporters followed by chemo-selective ligation of reporter groups was used for sequence-specific labeling of plasmid DNA (methyltransferase-directed Transfer of Activated Groups, mTAG). Although such a two-step procedure offers the flexibility of using one AdoMet analogue for labeling with different reporter groups, it adds an extra chemical step which is difficult to quantify on the DNA and can lead to partial DNA labeling. To overcome this limitation we have synthesized double-activated AdoMet analogues with over fiftyfold extended methyl group replacements, carrying different reporter or affinity groups in the activated side chain, and used them to sequence-specifically label plasmid and genomic DNA with DNA M Tases in one step. These enzymatic reactions are applied for genome barcoding and DNA mapping. In addition, we have developed a method to produce covalent DNA -protein conjugates by SNAP -tag technology. AdoMet analogues are used to enzymatically transfer benzylguanine residues to specific sites in DNA and the modified DNA is then reacted with SNAP -tag fusion proteins to specifically attach proteins to DNA . We have used this method to produce covalent plasmid DNA -antibody conjugates for targeted cell delivery.

This talk is part of the Keyser Group Seminar series.

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