title: Fluorophore-Conjugated Holliday Junctions for Generating Super-Bright Antibodies and Antibody Fragments.
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.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Zeyang
authors.initials: Z
authors.lastname: Li
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Guan-Yu
authors.initials: GY
authors.lastname: Chen
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Angelina M
authors.initials: AM
authors.lastname: Bilate
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Joao N
authors.initials: JN
authors.lastname: Duarte
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Ana M
authors.initials: AM
authors.lastname: Avalos
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142 (USA).
authors.forename: Tao
authors.initials: T
authors.lastname: Fang
authors.affiliation: Cell Signaling Technology, Beverly, MA 01915 (USA).
authors.forename: Roberto
authors.initials: R
authors.lastname: Barberena
authors.affiliation: Cell Signaling Technology, Beverly, MA 01915 (USA).
authors.forename: Shuji
authors.initials: S
authors.lastname: Sato
authors.affiliation: Department of Biology, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142 (USA). ploegh@wi.mit.edu.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000480:Alkynes / Q000737:chemistry*; D000906:Antibodies / Q000032:analysis* / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D045566:DNA, Cruciform / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*
chemical_list: D000480:Alkynes; D000906:Antibodies; D001386:Azides; D045566:DNA, Cruciform; D005456:Fluorescent Dyes
keywords: DNA technology; Holiday junctions; antibodies; imaging agents; immunocytochemistry