Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines

  • Bioconjug Chem. 2019 Jun 19;30(6):1773-1780. doi: 10.1021/acs.bioconjchem.9b00295.
Ulrike Reisacher  1 ,  Bastian Groitl  2 ,  Ralf Strasser  2 ,  Gergely B Cserép  3 ,  Péter Kele  3 ,  Hans-Achim Wagenknecht  1
Affiliations
  • 1. Karlsruhe Institute of Technology , Institute of Organic Chemistry , Fritz-Haber-Weg 6 , 76131 Karlsruhe , Germany.
  • 2. Dynamic Biosensors GmbH, Lochhamer Straße 15 , 82152 Martinsried , Germany.
  • 3. Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre for Natural Sciences , Hungarian Academy of Sciences , Magyar tudósok krt. 2 , 1117 Budapest , Hungary.
PMID: 31117344 DOI: 10.1021/acs.bioconjchem.9b00295
Abstract

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA Polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA Polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.

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