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

• Main Authors: Theile, Christopher S. (Author), Chen, Guan-Yu (Author), Bilate, Angelina M. (Author), Duarte, Joao N. (Author), Avalos, Ana M. (Author), Fang, Tao (Author), Barberena, Roberto (Author), Sato, Shuji (Author), Ploegh, Hidde (Contributor), Li, Zeyang,S.M.Massachusetts Institute of Technology (Author)
• Other Authors: Massachusetts Institute of Technology. Department of Biology (Contributor), Whitehead Institute for Biomedical Research (Contributor)
• Format: Article
• Language: English
• Published: Wiley Blackwell, 2017-05-01T18:47:33Z.
• Subjects: Article
• Online Access: Get fulltext

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.