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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...

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Autores principales: Yin, Maolu, Izadi, Manizheh, Tenglin, Karin, Viennet, Thibault, Zhai, Liting, Zheng, Ge, Arthanari, Haribabu, Dassama, Laura M. K., Orkin, Stuart H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
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.
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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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