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Molecules 2015, 20(11), 20805-20822; doi:10.3390/molecules201119723

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

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 & Ziegler Eurotope GmbH, Robert-Rössle-Str. 10, Berlin D-13125, Germany.
*
Author to whom correspondence should be addressed.
Academic Editor: Ramon Eritja
Received: 6 August 2015 / Revised: 3 November 2015 / Accepted: 10 November 2015 / Published: 23 November 2015
(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)
View Full-Text   |   Download PDF [3630 KB, uploaded 23 November 2015]   |  

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
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Kunkel, F.; Lurz, R.; Weinhold, E. A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI. Molecules 2015, 20, 20805-20822.

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