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.