Identification of the Binding Site between Aptamer sgc8c and PTK7,Analytical Chemistry

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Identification of the Binding Site between Aptamer sgc8c and PTK7
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-06-18 , DOI: 10.1021/acs.analchem.4c01186
Jianghuai Chen _{1,

2} , Jiaxuan He ₂ , Tao Bing ₂ , Yawei Feng _{1,

2} , Yifan Lyu _{1,

3} , Ming Lei ₄ , Weihong Tan _{1,

2}

Affiliation

Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets and have found broad applications in cancer early-stage detection, accurate drug delivery, and precise treatment. Although various aptamer screening methods have been developed over the past several decades, the accurate binding site between the target and the aptamer cannot be characterized during a typical aptamer screening process. In this research, we chose a widely used aptamer screened by our group, sgc8c, and its target protein tyrosine kinase 7 (PTK7) as the model aptamer and target and tried to determine the binding site between aptamer sgc8c and PTK7. Through sequential protein truncation, we confirmed that the exact binding site of sgc8c was within the region of Ig 3 to Ig 4 in the extracellular domain of PTK7. Using in vitro expressed Ig (3–4), we successfully acquired the crystal of an sgc8c-Ig (3–4) binding complex. The possible sgc8c-binding amino acid residues on PTK7 and PTK7-binding nucleotide residues on sgc8c were further identified and simulated by mass spectrometry and molecular dynamics simulation and finally verified by aptamer/protein truncation and mutation.

中文翻译：

适体 sgc8c 和 PTK7 之间结合位点的鉴定

适配体是能够特异性结合靶标的单链RNA或DNA分子，在癌症早期检测、精准药物输送和精准治疗等方面有着广泛的应用。尽管在过去的几十年里已经开发了各种适体筛选方法，但在典型的适体筛选过程中无法表征靶标与适体之间的准确结合位点。本研究选择本课题组筛选出的一种广泛使用的适配体sgc8c及其靶蛋白酪氨酸激酶7（PTK7）作为模型适配体和靶标，并试图确定适配体sgc8c与PTK7之间的结合位点。通过连续的蛋白质截断，我们证实sgc8c的确切结合位点位于PTK7胞外域的Ig 3至Ig 4区域内。利用体外表达的 Ig (3-4)，我们成功获得了 sgc8c-Ig (3-4) 结合复合物的晶体。通过质谱和分子动力学模拟进一步鉴定和模拟PTK7上可能的sgc8c结合氨基酸残基和sgc8c上PTK7结合核苷酸残基，并最终通过适体/蛋白质截短和突变进行验证。

更新日期：2024-06-18

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