Copper-free dual labeling of DNA by triazines and cyclopropenes as minimal orthogonal and bioorthogonal functions.

Two different and small functions for inverse electron demand Diels-Alder reactions were applied for dual labeling of DNA: the 1,2,4-triazine was attached to the 5-position of 2'-deoxyuridine triphosphate, and the 1-methylcyclopropene to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate. These two modified nucleotides were sequence-selectively incorporated into oligonucleotides by DNA polymerases. These products were labeled by two different fluorescent dyes using postsynthetic reactions that are not only bioorthogonal in general, but also mutually orthogonal.