Covalent protein-oligonucleotide conjugates by copper-free click reaction.
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.
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Santoshkumar L
SL
Khatwani
Jun Sung
JS
Kang
Daniel G
DG
Mullen
Michael A
MA
Hast
Lorena S
LS
Beese
Mark D
MD
Distefano
T Andrew
TA
Taton
D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D000480:Alkynes; D001386:Azides; D003300:Copper; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009841:Oligonucleotides; D011506:Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein