Novel Protein-oligonucleotide Conjugation Method Involving a High-affinity Capture HaloTag

Area of Science:

• Biochemistry
• Molecular Biology
• Medical Diagnostics

Background:

• Early disease diagnosis, including autoimmune diseases and cancer, relies on sensitive quantitative protein profiling.
• Conventional protein quantification methods often lack the required sensitivity for early-stage detection.

Purpose of the Study:

• To develop a highly sensitive protein quantification method using a modified protein-oligonucleotide conjugation technique.
• To establish HaloTag-mediated barcoding for enhanced protein molecule quantification.
• To demonstrate the preparation of protein-DNA complexes and detection of protein-protein interactions for antibody detection.

Main Methods:

• Developed a modified protein-oligonucleotide conjugation method termed HaloTag-mediated barcoding.
• Utilized click chemistry-based labeling for preparing HaloTag-barcoded proteins.
• Established protocols for ligand-oligonucleotide complex preparation, protein expression via plasmid DNA, and protein-oligonucleotide complex formation.

Main Results:

• Achieved higher sensitivity in protein quantification compared to conventional methods.
• Successfully prepared protein-DNA complexes and detected protein-protein interactions.
• Demonstrated the utility of the HaloTag protein barcode assay for antibody detection.

Conclusions:

• HaloTag-mediated barcoding offers a novel and efficient approach for highly sensitive protein quantification.
• The click chemistry-based protocol simplifies conjugation, avoiding additional purification steps.
• This method holds potential for advancing early disease diagnosis through improved protein profiling.