Site-specific one-pot dual labeling of DNA by orthogonal cycloaddition chemistry.
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.
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg, Germany.
Juliane
J
Schoch
Markus
M
Staudt
Ayan
A
Samanta
Manfred
M
Wiessler
Andres
A
Jäschke
D000480:Alkynes / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D057930:Click Chemistry; D003500:Cyclization; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D015394:Molecular Structure; D013194:Staining and Labeling / Q000379:methods*
D000480:Alkynes; D001386:Azides; D015342:DNA Probes; D004247:DNA