Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

  1. Jonathan CY Tang
  2. Eugene Drokhlyansky
  3. Behzad Etemad
  4. Stephanie Rudolph
  5. Binggege Guo
  6. Sui Wang
  7. Emily G Ellis
  8. Jonathan Z Li
  9. Constance L Cepko  Is a corresponding author
  1. Howard Hughes Medical Institute, Harvard Medical School, United States
  2. Harvard Medical School, United States

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 proteins.

Article and author information

Author details

  1. Jonathan CY Tang

    Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
    Competing interests
    Jonathan CY Tang, Submitted a patent application regarding destabilized nanobodies. International Application No. PCT/US2016/027749 Priority: US Prov. Appl. No. 62/148,595.
  2. Eugene Drokhlyansky

    Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
    Competing interests
    Eugene Drokhlyansky, Submitted a patent application regarding destabilized nanobodies. International Application No. PCT/US2016/027749Priority: US Prov. Appl. No. 62/148,595.
  3. Behzad Etemad

    Brigham and Women's Hospital, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  4. Stephanie Rudolph

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  5. Binggege Guo

    Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  6. Sui Wang

    Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
    Competing interests
    Sui Wang, Submitted a patent application regarding destabilized nanobodies. International Application No. PCT/US2016/027749Priority: US Prov. Appl. No. 62/148,595.
  7. Emily G Ellis

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  8. Jonathan Z Li

    Brigham and Women's Hospital, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  9. Constance L Cepko

    Departments of Genetics and Ophthalmology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States
    For correspondence
    cepko@genetics.med.harvard.edu
    Competing interests
    Constance L Cepko, Submitted a patent application regarding destabilized nanobodies. International Application No. PCT/US2016/027749 Priority: US Prov. Appl. No. 62/148,595.

Reviewing Editor

  1. Liqun Luo, Howard Hughes Medical Institute, Stanford University, United States

Ethics

Animal experimentation: The Institutional Animal Care and Use Committee at Harvard Medical School approved all animal experiments conducted under protocols 428-R98, 04537 and 1493.

Version history

  1. Received: February 17, 2016
  2. Accepted: May 19, 2016
  3. Accepted Manuscript published: May 20, 2016 (version 1)
  4. Version of Record published: June 27, 2016 (version 2)

Copyright

© 2016, Tang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jonathan CY Tang
  2. Eugene Drokhlyansky
  3. Behzad Etemad
  4. Stephanie Rudolph
  5. Binggege Guo
  6. Sui Wang
  7. Emily G Ellis
  8. Jonathan Z Li
  9. Constance L Cepko
(2016)
Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies
eLife 5:e15312.
https://doi.org/10.7554/eLife.15312

Share this article

https://doi.org/10.7554/eLife.15312

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