Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
Photocrosslinking of cDNA Display Molecules with Their Target Proteins as a New Strategy for Peptide Selection
复制标题
DOI：
10.3390/molecules25061472
复制
发表时间：
2020-03
期刊：
影响因子：
4.6
通讯作者：
Takuya Terai;T. Koike;N. Nemoto
中科院分区：
文献类型：
--
作者：
Takuya Terai;T. Koike;N. Nemoto
在药物发现和化学生物学研究期间，通常在体外选择针对给定靶分子的结合肽。在用于此目的的几种展示技术中，互补DNA（cDNA）展示（通过特别设计的嘌呤霉素缀合的DNA连接的肽及其编码cDNA的共价复合物）在文库大小、化学稳定性和修饰的灵活性方面是独特的。然而，cDNA展示文库的选择经常遭受来自非特异性结合的假阳性。虽然严格洗涤是一种简单的解决方案，但这也导致具有中等亲和力的特异性结合剂的损失，因为相互作用是非共价的。为了解决这个问题，在此，我们提出了一种方法，共价连接cDNA展示分子与它们的靶蛋白，使用光照射。我们设计了一种含有光交联核酸的嘌呤霉素DNA接头，并利用该接头制备了cDNA展示分子。靶蛋白也用短单链DNA标记，其应与接头瞬时杂交。在紫外线（UV）光照射下，编码正确肽适体的cDNA展示分子与溶液中的靶蛋白形成稳定的交联产物，而编码对照肽的展示分子则没有。虽然进一步的优化和改进是必要的，结果铺平了道路，有效地选择肽适体在多分子拥挤的生物系统。
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.