Covalent protein-oligonucleotide conjugates by copper-free click reaction

Santoshkumar L. Khatwani, Jun Sung Kang, Daniel G. Mullen, Michael A. Hast, Lorena S. Beese, Mark D Distefano, T. A Taton

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38 Scopus citations


Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates - where the connection between the two components is at a defined location in both the protein and the ODN - under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were 'clicked' to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.

Original languageEnglish (US)
Pages (from-to)4532-4539
Number of pages8
JournalBioorganic and Medicinal Chemistry
Issue number14
StatePublished - Jul 15 2012

Bibliographical note

Funding Information:
We thank Jonathan Dozier for providing 1-azido-6,7-dihydrogeranyl diphosphate 3 (C 10 ), James Wollack for 1-azido-10,11-dihydrofarnesyl diphosphate 3 (C 15 ), Professor Karin Musier-Forsyth for helpful discussions concerning NC protein, and Professor George Barany for use of his automated peptide synthesizer. This work was supported by the NIH ( CA122603 and GM084152 ).


  • Bioconjugation
  • Click chemistry
  • Protein immobilization
  • Protein prenylation
  • Protein-oligodeoxynucleotide conjugates


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