A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaIReportar como inadecuado




A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

1

Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, Aachen D-52056, Germany

2

Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, Berlin D-14195, Germany



Present address: Eckert and Ziegler Eurotope GmbH, Robert-Rössle-Str. 10, Berlin D-13125, Germany.





*

Author to whom correspondence should be addressed.



Academic Editor: Ramon Eritja

Abstract DNA methyltransferases MTases catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine AdoMet or SAM to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus M.TaqI is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5′-TCGA-3′ sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus M.HhaI. Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5′-GCGC-3′ sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA. View Full-Text

Keywords: S-adenosyl-l-methionine; AdoMet; aziridine; CpG methylation; cofactor; 7-deazaadenosine derivative; DNA methyltransferase; DNA methylation; DNA labeling; Sonogashira coupling; SAM S-adenosyl-l-methionine; AdoMet; aziridine; CpG methylation; cofactor; 7-deazaadenosine derivative; DNA methyltransferase; DNA methylation; DNA labeling; Sonogashira coupling; SAM





Autor: Falk Kunkel 1,†, Rudi Lurz 2 and Elmar Weinhold 1,*

Fuente: http://mdpi.com/



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