title: A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.
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.
authors.affiliation: Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, Aachen D-52056, Germany. Falk_Schmidt@gmx.de.
authors.forename: Falk
authors.initials: F
authors.lastname: Kunkel
authors.affiliation: Max-Planck-Institute for Molecular Genetics, Ihnestrasse 73, Berlin D-14195, Germany. Lurz@molgen.mpg.de.
authors.forename: Rudi
authors.initials: R
authors.lastname: Lurz
authors.affiliation: Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, Aachen D-52056, Germany. elmar.weinhold@oc.rwth-aachen.de.
authors.forename: Elmar
authors.initials: E
authors.lastname: Weinhold
mesh_terms: D001388:Aziridines / Q000138:chemical synthesis / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002384:Catalysis; D018899:CpG Islands; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry / Q000648:ultrastructure; D011485:Protein Binding; D014372:Tubercidin / Q000737:chemistry*
chemical_list: D001388:Aziridines; D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D014372:Tubercidin
keywords: 7-deazaadenosine derivative; AdoMet; CpG methylation; DNA labeling; DNA methylation; DNA methyltransferase; S-adenosyl-l-methionine; SAM; Sonogashira coupling; aziridine; cofactor