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Evolution of nanobodies specific for BCL11A
Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportu...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
National Academy of Sciences
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933118/ https://www.ncbi.nlm.nih.gov/pubmed/36626555 http://dx.doi.org/10.1073/pnas.2218959120 |
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author | Yin, Maolu Izadi, Manizheh Tenglin, Karin Viennet, Thibault Zhai, Liting Zheng, Ge Arthanari, Haribabu Dassama, Laura M. K. Orkin, Stuart H. |
author_facet | Yin, Maolu Izadi, Manizheh Tenglin, Karin Viennet, Thibault Zhai, Liting Zheng, Ge Arthanari, Haribabu Dassama, Laura M. K. Orkin, Stuart H. |
author_sort | Yin, Maolu |
collection | PubMed |
description | Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest. |
format | Online Article Text |
id | pubmed-9933118 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | National Academy of Sciences |
record_format | MEDLINE/PubMed |
spelling | pubmed-99331182023-02-17 Evolution of nanobodies specific for BCL11A Yin, Maolu Izadi, Manizheh Tenglin, Karin Viennet, Thibault Zhai, Liting Zheng, Ge Arthanari, Haribabu Dassama, Laura M. K. Orkin, Stuart H. Proc Natl Acad Sci U S A Biological Sciences Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Here, we describe the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. We obtained first-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) from a synthetic yeast surface display library, and employed error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA-binding specificity. Given the ease of manipulation of nanobodies and their exquisite specificity, nanobody-mediated TPD of TFs should be suitable for dissecting regulatory relationships of TFs and gene targets and validating therapeutic potential of proteins of interest. National Academy of Sciences 2023-01-10 2023-01-17 /pmc/articles/PMC9933118/ /pubmed/36626555 http://dx.doi.org/10.1073/pnas.2218959120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) . |
spellingShingle | Biological Sciences Yin, Maolu Izadi, Manizheh Tenglin, Karin Viennet, Thibault Zhai, Liting Zheng, Ge Arthanari, Haribabu Dassama, Laura M. K. Orkin, Stuart H. Evolution of nanobodies specific for BCL11A |
title | Evolution of nanobodies specific for BCL11A |
title_full | Evolution of nanobodies specific for BCL11A |
title_fullStr | Evolution of nanobodies specific for BCL11A |
title_full_unstemmed | Evolution of nanobodies specific for BCL11A |
title_short | Evolution of nanobodies specific for BCL11A |
title_sort | evolution of nanobodies specific for bcl11a |
topic | Biological Sciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933118/ https://www.ncbi.nlm.nih.gov/pubmed/36626555 http://dx.doi.org/10.1073/pnas.2218959120 |
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