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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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. |
format | Online Article Text |
id | pubmed-7146492 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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