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Updated: May 21, 2026

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes
Published on: July 6, 2016
Site-specific one-pot dual labeling of DNA by orthogonal cycloaddition chemistry
Juliane Schoch1, Markus Staudt, Ayan Samanta
1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany.
Abstract:
Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.
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