Covalent protein-oligonucleotide conjugates by copper-free click reaction

Area of Science:

• Bioconjugation Chemistry
• Molecular Biology
• Materials Science

Background:

• Synthesizing discrete protein-oligodeoxynucleotide (protein-ODN) conjugates with defined linkages is challenging.
• Mild reaction conditions and high yields are desirable for biological applications.
• Existing methods like copper-catalyzed azide-alkyne cycloaddition (CuAAC) require a metal catalyst.

Purpose of the Study:

• To demonstrate a novel strategy for synthesizing discrete protein-ODN conjugates.
• To utilize strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC) for copper-free bioconjugation.
• To establish SPAAC as a versatile method for protein-ODN conjugation and surface immobilization.

Main Methods:

• Enzymatic prenylation of proteins with azide-functionalized tags.
• Modification of oligodeoxynucleotides (ODNs) with dibenzocyclooctyne (DIBO).
• Strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC) for bioconjugation.
• Purification and characterization using size-exclusion chromatography and gel electrophoresis.

Main Results:

• Successful synthesis of discrete protein-ODN conjugates via SPAAC.
• SPAAC reaction yields and times are comparable to CuAAC.
• SPAAC proceeds efficiently without a copper catalyst.
• Demonstrated immobilization of azide-modified proteins onto surfaces using DIBO-ODN linkers.

Conclusions:

• SPAAC provides a simple, efficient, and catalyst-free method for synthesizing discrete protein-ODN conjugates.
• This copper-free click chemistry is a versatile alternative to traditional CuAAC methods.
• The developed strategy facilitates protein-ODN conjugation and surface functionalization for various biological applications.