Sequence-Specific DNA Labeling Using Methyltransferases

Pljevaljčić, Goran; Schmidt, Falk; Peschlow, Alexander; Weinhold, Elmar G.

doi:10.1385/1-59259-813-7:145

Cited by 10 publications

(13 citation statements)

References 11 publications

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“…In the case of the Weinhold’s approach, instead of S-adenosyl methionine, an artificial fluorescent substrate with the chemically active aziridine group was used. Thus, instead of methyl group, a fluorescent label can be inserted into DNA target sequence [36,37,38]. However, this method is too far from the in vivo applications because it requires the presence of methylases of desired specificity in cells, as well as a synthetic chemical substrate that can be cytotoxic.…”

Section: Probes For Double-stranded Dna Imaging By Fluorescencementioning

confidence: 99%

Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

et al. 2013

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This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i) sequence-specific peptides and proteins; (ii) triplex-forming oligonucleotides and (iii) polyamide oligo(N-methylpyrrole/N-methylimidazole) minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, OPEN ACCESSMolecules 2013, 18 15358 molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

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Section: Probes For Double-stranded Dna Imaging By Fluorescencementioning

confidence: 99%

Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

et al. 2013

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“…The enzymes are tricked into catalysing a nucleophilic aziridine ring opening reaction instead of a methyl group transfer, and couple the whole aziridine cofactor to their target nucleobase in DNA [28]. By attaching chemical groups to different positions of the adenine ring the aziridine cofactors work in combination with DNA MTases as delivery systems for reporter groups such as fluorophores [29] or biotin [30,31]. Thus this one-step method was termed SMILing DNA (sequence-specific methyltransferaseinduced labelling of DNA) [32] and used to study cell transfection with fluorescently labelled DNA [33] or to nanostructure DNA and position gold nanoparticles on DNA with biotinylated DNA [34,35].…”

Section: Dna Mtases (Dna Methyltransferases)mentioning

confidence: 99%

Sequence-specific covalent labelling of DNA

Gottfried

Weinhold

2011

Biochemical Society Transactions

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Sequence-specific DNA modification is of significance for applications in bio- and nano-technology, medical diagnostics and fundamental life sciences research. Preferentially, labelling should be performed covalently, which avoids doubts about label dissociation from the DNA under various conditions. Several methods to label native DNA have been developed in the last two decades. Triple-helix-forming oligodeoxynucleotides and hairpin polyamides that bind DNA sequences specifically in the major and minor groove respectively were used as targeting devices for subsequent covalent labelling. In addition, enzyme-directed labelling approaches utilizing nicking endonucleases in combination with DNA polymerases or DNA methyltransferases have been employed. This review summarizes various techniques useful for functionalization of long native DNA.

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“…This was achieved by the direct attachment of different reporter groups (biotin and fluorophores) to the adenine ring of N-adenosylaziridine (43) via a flexible linker (Scheme 9.7B). DNA MTase-mediated coupling with long plasmid DNA occurs in quantitative manner and is also sequence-specific [169][170][171].…”

Section: Aziridine Analogues Of Adometmentioning

confidence: 99%

S‐Adenosyl‐L‐Methionine and Related Compounds

Dalhoff¹,

Weinhold

2008

Modified Nucleosides

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The sulfonium compound S-adenosyl-L-methionine (AdoMet, also known as SAM or SAMe, 1) is one of the most versatile biomolecules in nature and one of the most widely used enzyme substrates, second perhaps only to adenosine triphosphate (ATP) (Schemes 9.1 and 9.2). AdoMet (1) may be found in all phyla of life, and is one of the molecules crucial for the existence of living organisms. First identified by Cantoni in 1953 [1, 2] as the active methyl donor formed from L-methionine and ATP, AdoMet (1) proved to be not only the major methyl donor in nature but also to be involved in many other metabolic reactions [3,4]. The versatility of the cofactor is based on the reactivity of the pivotal sulfonium center, which activates the adjacent carbon atoms for nucleophilic attack, deprotonation, and radical formation. This leads to a unique cofactor in which all constituents are used in biochemical reactions. The group transfer reactions result in the release of one of three different thioether products which are more stable than the sulfonium compound, and thereby providing the driving force for these enzyme-catalyzed reactions. These highly preferable thermodynamics of AdoMet-dependent transfer reactions result in a strong preference for this cofactor compared, for example, to other methyl donors such as N5-methyltetrahydrofolate. Due to the electron lone pair in addition to the three different substituents, the sulfonium group is chiral, and the naturally occurring AdoMet (1) is S-configured at sulfur [5].Many other naturally occurring alkylated thioadenosines are derived from AdoMet (1) such as S-adenosyl-L-homocysteine (AdoHcy, also called SAH, 2) and 5 0 -deoxy-5 0 -methylthioadenosine (MTA, 3) (Scheme 9.3), although these compounds and their related 5 0 -thioethers are beyond the scope of this chapter. AdoMet (1) and its derivatives are involved in key metabolic reactions, cell cycle regulation and the storage of epigenetic information, which makes them not only highly interesting for studying biological pathways but also as in-vitro or in-vivo biochemical tools and candidates for diagnostics and therapeutics.

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Sequence-Specific DNA Labeling Using Methyltransferases

Cited by 10 publications

References 11 publications

Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

Sequence-specific covalent labelling of DNA

S‐Adenosyl‐L‐Methionine and Related Compounds

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