title: Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.
abstract: The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.|Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
authors.forename: Jonathan Cy
authors.identifier: 0000-0002-2376-6901
authors.initials: JC
authors.lastname: Tang
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.|Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
authors.forename: Eugene
authors.initials: E
authors.lastname: Drokhlyansky
authors.affiliation: Brigham and Women's Hospital, Harvard Medical School, Boston, United States.
authors.forename: Behzad
authors.initials: B
authors.lastname: Etemad
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Rudolph
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.|Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
authors.forename: Binggege
authors.initials: B
authors.lastname: Guo
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.|Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
authors.forename: Sui
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Ellis
authors.affiliation: Brigham and Women's Hospital, Harvard Medical School, Boston, United States.
authors.forename: Jonathan Z
authors.initials: JZ
authors.lastname: Li
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.|Department of Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
authors.forename: Constance L
authors.identifier: 0000-0002-9945-6387
authors.initials: CL
authors.lastname: Cepko
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism*; D003584:Cytological Techniques / Q000379:methods*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D061905:Single-Domain Antibodies / Q000378:metabolism*
chemical_list: D000941:Antigens; D061905:Single-Domain Antibodies
keywords: GFP; HIV-1; antibody engineering; developmental biology; human; mouse; nanobodies; neuroscience; stem cells