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Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection

Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed pu...

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Detalles Bibliográficos
Autores principales: Terai, Takuya, Koike, Tomoyuki, Nemoto, Naoto
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146492/
https://www.ncbi.nlm.nih.gov/pubmed/32214008
http://dx.doi.org/10.3390/molecules25061472
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author Terai, Takuya
Koike, Tomoyuki
Nemoto, Naoto
author_facet Terai, Takuya
Koike, Tomoyuki
Nemoto, Naoto
author_sort Terai, Takuya
collection PubMed
description Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems.
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spelling pubmed-71464922020-04-20 Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection Terai, Takuya Koike, Tomoyuki Nemoto, Naoto Molecules Article Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding. Although rigorous washing is a straightforward solution, this also leads to the loss of specific binders with moderate affinity because the interaction is non-covalent. To address this issue, herein, we propose a method to covalently link cDNA display molecules with their target proteins using light irradiation. We designed a new puromycin DNA linker that contains a photocrosslinking nucleic acid and prepared cDNA display molecules using the linker. Target proteins were also labeled with a short single-stranded DNA that should transiently hybridize with the linker. Upon ultraviolet (UV) light irradiation, cDNA display molecules encoding correct peptide aptamers made stable crosslinked products with the target proteins in solution, while display molecules encoding control peptides did not. Although further optimization and improvement is necessary, the results pave the way for efficient selection of peptide aptamers in multimolecular crowding biosystems. MDPI 2020-03-24 /pmc/articles/PMC7146492/ /pubmed/32214008 http://dx.doi.org/10.3390/molecules25061472 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Terai, Takuya
Koike, Tomoyuki
Nemoto, Naoto
Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title_full Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title_fullStr Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title_full_unstemmed Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title_short Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
title_sort photocrosslinking of cdna display molecules with their target proteins as a new strategy for peptide selection
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146492/
https://www.ncbi.nlm.nih.gov/pubmed/32214008
http://dx.doi.org/10.3390/molecules25061472
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