A Platform for Site-Specific DNA-Antibody Bioconjugation by Using Benzoylacrylic-Labelled Oligonucleotides

Area of Science:

• Bioconjugation Chemistry
• Molecular Biology
• Biochemistry

Background:

• Current DNA-antibody bioconjugation methods face challenges with site-specificity, stoichiometry, and conjugate stability.
• These limitations hinder the effective application of DNA-antibody conjugates in biological systems.

Purpose of the Study:

• To develop a novel, versatile platform for creating site-specific DNA-antibody bioconjugates.
• To overcome the limitations of existing bioconjugation strategies for DNA and antibodies.

Main Methods:

• Utilized a benzoylacrylic acid pentafluorophenyl ester reagent for labeling DNA oligonucleotides.
• Performed site-specific conjugation of labeled oligonucleotides to proteins and antibodies via cysteine residues.
• Employed a novel liquid chromatography-mass spectrometry (LC-MS) protocol for homogeneity assessment.
• Applied the resulting bioconjugates in fluorescence and super-resolution microscopy for cell imaging.

Main Results:

• Successfully prepared homogeneous DNA-antibody bioconjugates with high site-specificity.
• Demonstrated the stability of the bioconjugates under physiological conditions.
• Validated the utility of the bioconjugates in advanced cell imaging techniques.

Conclusions:

• The developed benzoylacrylic acid reagent offers a robust and versatile platform for DNA-antibody bioconjugation.
• This method yields site-specific, well-defined, and plasma-stable bioconjugates suitable for biological applications.
• The bioconjugates are effective tools for cellular imaging, showcasing potential in diagnostics and research.