Triazine-modified 7-deaza-2'-deoxyadenosines are better suited for bioorthogonal labelling of DNA by PCR than 2'-deoxyuridines.

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labelling 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 multilabelled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 1-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 labelling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.