Sequence-specific Methyltransferase-Induced Labelling (SMILing) of plasmid DNA for studying cell transfection

Schmidt, Falk; Hüben, Michael; Gider, Basar; Renault, F; Teulade-Fichou, Marie-Paule; Weinhold, Elmar G.

doi:10.1016/j.bmc.2007.04.054

Cited by 28 publications

(21 citation statements)

References 50 publications

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“…These analogs work well in vitro and in vivo , and can modify the substrates with the anchor groups instead of the methyl groups in an enzyme-specific manner. By using this technology, DNAs (Schmidt et al, 2008), RNAs (Motorin et al, 2010), and proteins (Peters et al, 2010) have been successfully labeled with an alkyne or an azide. Similar enzymatic labeling methods for protein substrates have also been studied for other post-translational modifications, including phosphorylation (Lee et al, 2009) and acetylation (Yang et al, 2010).…”

Section: Novel Labeling Methods and The Use Of Click Chemistry In Quamentioning

confidence: 99%

Specific and quantitative labeling of biomolecules using click chemistry

Horisawa

2014

Front. Physiol.

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Specific and highly efficient fluorescent labeling techniques for biomolecules, especially for proteins, are required for the quantitative analyses of bio-phenomena and for subsequent systems biology. Although expression of exogenous proteins fused with fluorescent tags, such as green fluorescent protein, is the most widely used method for quantitative bio-analysis, the following problems need to be considered carefully: (1) precise stoichiometric control in living cells is difficult, and (2) the bulkiness of the fluorescent tags restricts analysis of the inherent physical and biological properties of the proteins. Therefore, novel techniques to specifically and stoichiometrically label intrinsic proteins or other biomolecules in living cells should be developed. Click chemistry reactions (e.g., Huisgen cycloaddition and Staudinger ligation) are the most promising approaches for this purpose, because these chemical reactions have following advantages: (1) bioorthogonal reactions; (2) mild reaction conditions suitable for fragile biomolecules, cells, and tissues; (3) extremely high reaction ratio; (4) small size of the functional groups for the cross-coupling reactions; (5) stable covalent bonding; and (6) simple metabolic labeling procedures in living cells, using various biomolecular analogs. Diverse quantitative biological studies have been carried out using this technology (e.g., quantification of novel synthesized proteins and observation of post-translational modifications). In this review, I explain the basics of chemical probing with click chemistry, and discuss its recent applications in the field of quantitative biology. Furthermore, I discuss the capability, significance, and future of the chemical probing of proteins, with an emphasis on the use of click chemistry in the field of the quantitative biology.

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Section: Novel Labeling Methods and The Use Of Click Chemistry In Quamentioning

confidence: 99%

Specific and quantitative labeling of biomolecules using click chemistry

Horisawa

2014

Front. Physiol.

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“…In an example from the Weinhold group, Cy3 dyes were covalently coupled to pUC19 and pBR322 plasmids using an N‐adenosylaziridine cofactor 51. In this study, the labeled plasmids were successfully transfected into CHO‐k1 cells.…”

Section: Current and Future Applicationsmentioning

confidence: 99%

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

et al. 2017

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Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S‐adenosyl‐l‐Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly‐activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet‐dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology.

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“…Sequence specific labeling of DNA and RNA with AdoMet analogues is of major interest for genotyping (DNA mapping) 36 and methylation detection 39 , functional studies of DNA/RNA and DNA/RNA-modifying enzymes 15 as well as for (nano)biotechnology 13,14 and gene delivery 19 . Other methods for sequence specific covalent DNA labeling rely on synthetic triple helix-forming oligodeoxynucleotides, peptide nucleic acids or hairpin polyamides as targeting devices or on sequence specific nicking endonucleases (NEases) followed by nick translation labeling.…”

Section: Double Activated Cofactormentioning

confidence: 99%

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

Hanz

Jung

Giesbertz

et al. 2014

JoVE

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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

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Sequence-specific Methyltransferase-Induced Labelling (SMILing) of plasmid DNA for studying cell transfection

Cited by 28 publications

References 50 publications

Specific and quantitative labeling of biomolecules using click chemistry

Specific and quantitative labeling of biomolecules using click chemistry

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

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

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