title: Nucleotidyl transferase assisted DNA labeling with different click chemistries.
abstract: Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.
authors.affiliation: Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
authors.forename: Marie-Luise
authors.initials: ML
authors.lastname: Winz
authors.affiliation: Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
authors.forename: Eva Christina
authors.initials: EC
authors.lastname: Linder
authors.affiliation: Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
authors.forename: Timon
authors.initials: T
authors.lastname: André
authors.affiliation: Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Becker
authors.affiliation: Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany jaeschke@uni-hd.de.
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
mesh_terms: D001386:Azides / Q000737:chemistry; D057930:Click Chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase; D009711:Nucleotides / Q000378:metabolism
chemical_list: D001386:Azides; D009711:Nucleotides; D004247:DNA; D004253:DNA Nucleotidylexotransferase