Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan
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Academic Editors: Daisuke Miyoshi, Akio Ojida and Kazuhito Tabata
Molecules 2020, 25(6), 1472; https://doi.org/10.3390/molecules25061472
Received: 28 January 2020
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Revised: 19 March 2020
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Accepted: 23 March 2020
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Published: 24 March 2020
(This article belongs to the Special Issue Structure, Interaction, Reaction, and Function of Biomolecules in Multimolecular Crowding Biosystems)
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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Keywords:
directed evolution; in vitro selection; multimolecular crowding biosystems; cDNA display; photocrosslinker; peptide aptamer
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MDPI and ACS Style
Terai, T.; Koike, T.; Nemoto, N. Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection. Molecules 2020, 25, 1472. https://doi.org/10.3390/molecules25061472
AMA Style
Terai T, Koike T, Nemoto N. Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection. Molecules. 2020; 25(6):1472. https://doi.org/10.3390/molecules25061472
Chicago/Turabian StyleTerai, Takuya, Tomoyuki Koike, and Naoto Nemoto. 2020. "Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection" Molecules 25, no. 6: 1472. https://doi.org/10.3390/molecules25061472
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