HaloTag-based conjugation of proteins to barcoding-oligonucleotides

Junshi Yazaki, Yusuke Kawashima, Taisaku Ogawa, Atsuo Kobayashi, Mayu Okoshi, Takashi Watanabe, Suguru Yoshida, Isao Kii, Shohei Egami, Masayuki Amagai, Takamitsu Hosoya, Katsuyuki Shiroguchi, Osamu Ohara

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Abstract

Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.

Original languageEnglish
Pages (from-to)e8
JournalNucleic acids research
Volume48
Issue number2
DOIs
Publication statusPublished - 2020 Jan 24

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ASJC Scopus subject areas

  • Genetics

Cite this

Yazaki, J., Kawashima, Y., Ogawa, T., Kobayashi, A., Okoshi, M., Watanabe, T., Yoshida, S., Kii, I., Egami, S., Amagai, M., Hosoya, T., Shiroguchi, K., & Ohara, O. (2020). HaloTag-based conjugation of proteins to barcoding-oligonucleotides. Nucleic acids research, 48(2), e8. https://doi.org/10.1093/nar/gkz1086