Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan.
Molecules. 2020 Mar 24;25(6):1472. doi: 10.3390/molecules25061472.
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.
在药物发现和化学生物学研究中，经常在体外选择针对特定靶分子的结合肽。在几种用于此目的的展示技术中，互补 DNA（cDNA）展示（通过专门设计的与嘌呤霉素缀合的 DNA 连接的肽与其编码 cDNA 的共价复合物）在文库大小、化学稳定性和修饰灵活性方面具有独特性。然而，cDNA 展示文库的选择经常受到来自非特异性结合的假阳性的影响。虽然严格洗涤是一种直接的解决方案，但这也会导致中等亲和力的特异性结合物的损失，因为这种相互作用是非共价的。为了解决这个问题，我们在此提出了一种使用光照射将 cDNA 展示分子与其靶蛋白共价连接的方法。我们设计了一种新的嘌呤霉素 DNA 接头，其中包含光交联核酸，并使用该接头制备 cDNA 展示分子。目标蛋白也用短的单链 DNA 标记，该 DNA 应该与接头瞬时杂交。在紫外线（UV）照射下，编码正确肽适体的 cDNA 显示分子在溶液中与靶蛋白形成稳定的交联产物，而编码对照肽的显示分子则没有。尽管还需要进一步的优化和改进，但这些结果为在多分子拥挤生物系统中有效选择肽适体铺平了道路。
