Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Abstract

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection. Video LinkThe video component of this article can be found at

Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores

“…We modify short sequence motifs with a biotin residue by using a DNA-methyltransferase (MTase) and a synthetic cofactor analogue. 18 The biotin sites are then labeled by tight binding of monovalent streptavidin 19 thereby creating additional, sequence specific blockades as the DNA passes through the solid-state nanopore. We demonstrate the feasibility of our method by mapping a variety of DNA plasmid and bacteriophage genomes and find a minimum resolvable distance between sequence motifs of ∼200 bp.…”

Bacteriophage strain typing by rapid single molecule analysis

“…To generate sequence specific labelling we used the DNA MTase M.TaqI to catalyze the transfer of a carboxytetramethylrhodamine (TAMRA) fluorophore from the synthetic cofactor AdoYnTAMRA onto the adenine residue within its TCGA recognition sequence ( 32 , 33 ). A total of 1 μg of 48 kbp λ-DNA containing 121 M.TaqI sites or 39 kbp T7 DNA containing 111 M.TaqI sites (both from New England Biolabs, Ipswich MA, USA), was treated with 1.1 μg of M.TaqI and 40 μM of AdoYnTAMRA in labelling buffer (20 mM Tris/HOAc, 10 mM Mg(Cl) 2 , 50 mM KOAc, 1 mM DTT, pH 7.9) in a total reaction volume of 25 μl at 37°C for 1 h (Scheme 1 ).…”

Covalent labeling of nucleic acids

“…10B, 11). 153 In 2007, the groups of Klimašauskas and Weinhold demonstrated sequence-specific labeling of oligonucleotides by functionalizing methylation sites of DNA with an amino group. Plasmid DNA was modified using an AdoMet analog with an amino-alkyl group within the side chain using the enzymes M.TaqI or M.HhaI.…”