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.
Bibliographical noteFunding 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 ).
- Click chemistry
- Protein immobilization
- Protein prenylation
- Protein-oligodeoxynucleotide conjugates