Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments

Zeyang Li, Christopher S. Theile, Guan-Yu Chen, Angelina M. Bilate, Joao N. Duarte, Ana M. Avalos, Tao Fang, Roberto Barberena, Shuji Sato, Hidde L. Ploegh

Research output: Contribution to journalArticle

19 Scopus citations

Abstract

The site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching from multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified fluorescent single-domain antibody fragment or an intact immunoglobulin produced in a sortase-catalyzed reaction. These fluorescent Holliday junctions improve fluorescence yields for both single-domain and full-sized antibodies without deleterious effects on antigen binding. Many hands make light work: Holliday junctions with fluorophores (red) conjugated at three of the four arms can be used to attach multiple fluorophores to both single-domain and full-sized antibodies (blue) with sufficient spacing to avoid self-quenching. The resulting conjugates showed improved fluorescence yields over those of singly fluorophore-conjugated antibodies without adversely affecting antigen binding.

Original languageEnglish
Pages (from-to)11706-11710
Number of pages5
JournalAngewandte Chemie - International Edition
Volume54
Issue number40
DOIs
StatePublished - 1 Sep 2015

Keywords

  • DNA technology
  • Holiday junctions
  • antibodies
  • imaging agents
  • immunocytochemistry

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    Li, Z., Theile, C. S., Chen, G-Y., Bilate, A. M., Duarte, J. N., Avalos, A. M., Fang, T., Barberena, R., Sato, S., & Ploegh, H. L. (2015). Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments. Angewandte Chemie - International Edition, 54(40), 11706-11710. https://doi.org/10.1002/anie.201505277