The Institute of Chemistry, The Centre of Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem91904, Israel.
J Phys Chem B. 2022 Nov 10;126(44):8931-8939. doi: 10.1021/acs.jpcb.2c05624. Epub 2022 Oct 31.
A method to computationally and experimentally identify aptamers against short peptides or amino acid clusters is introduced. The method involves the selection of a well-defined protein aptamer complex and the extraction of the peptide sequence participating in the binding of the protein to the aptamer. The subsequent fragmentation of the peptide sequence into short peptides and the in silico docking-guided identification of affinity complexes between the miniaturized peptides and the antiprotein aptamer, followed by experimental validation of the binding features of the short peptides with the antiprotein aptamers, leads to the identification of new short peptide-aptamer complexes. This is exemplified with the identification of the pentapeptide RYERN as the scaffold that binds thrombin to the DNA thrombin aptamer (DNA TA). In silico docking studies followed by microscale thermophoresis (MST) experiments demonstrate that the miniaturized tripeptides RYE, YER, and ERN reveal selective binding affinities toward the DNA TA. In addition, docking and MST experiments show that the ribonucleotide-translated RNA TA shows related binding affinities of YER to the DNA TA. Most importantly, we demonstrate that the separated amino acids Y/E/R assemble as a three amino acid cluster on the DNA TA and RNA TA aptamers in spatial configurations similar to the tripeptide YER on the respective aptamers. The clustering phenomenon is selective for the YER tripeptide system. The method to identify binding affinities of miniaturized peptides to known antiprotein aptamers and the specific clustering of single amino acids on the aptamers is further demonstrated by in silico and experimental identification of the binding of the tripeptide RET and the selective clustering of the separated amino acids R/E/T onto a derivative of the AS1411 aptamer against the nucleolin receptor protein.
介绍了一种计算和实验鉴定针对短肽或氨基酸簇的适体的方法。该方法涉及选择一个定义明确的蛋白质适体复合物，并提取参与蛋白质与适体结合的肽序列。随后将肽序列碎片化成长肽，并通过计算机对接指导鉴定小分子肽与反蛋白适体之间的亲和复合物，然后通过实验验证短肽与反蛋白适体的结合特征，从而鉴定新的短肽-适体复合物。这在鉴定五肽 RYERN 作为将凝血酶结合到 DNA 凝血酶适体（DNA TA）的支架方面得到了例证。计算机对接研究随后进行微量热泳动（MST）实验表明，小型化的三肽 RYE、YER 和 ERN 对 DNA TA 表现出选择性结合亲和力。此外，对接和 MST 实验表明，翻译后的核糖核苷酸 RNA TA 对 DNA TA 表现出与 YER 相关的结合亲和力。最重要的是，我们证明在 DNA TA 和 RNA TA 适体上，分离的氨基酸 Y/E/R 以类似于三肽 YER 在各自适体上的空间构象组装成一个三氨基酸簇。聚类现象对 YER 三肽系统是选择性的。通过鉴定小型肽与已知反蛋白适体的结合亲和力以及在适体上单个氨基酸的特异性聚类的计算机和实验鉴定，进一步证明了这种方法，通过鉴定三肽 RET 的结合以及分离的氨基酸 R/E/T 选择性聚类到针对核仁蛋白受体蛋白的 AS1411 适体衍生物上，进一步证明了这种方法。
