Identification of the Binding Site between Aptamer sgc8c and PTK7

作者：Jianghuai Chen, Jiaxuan He, Tao Bing, Yawei Feng, Yifan Lyu, Ming Lei, Weihong Tan · 发表于：Analytical Chemistry · 年份：2024 · DOI：10.1021/acs.analchem.4c01186 · 被引用次数：16 · 研究领域：Advanced biosensing and bioanalysis techniques、RNA and protein synthesis mechanisms、RNA Interference and Gene Delivery

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.