DNA适体因其对蛋白质靶点的高结合亲和力和特异性而成为疾病治疗诊断学中的新型分子工具，而蛋白质靶点依赖于它们折叠成独特的三维（3D）结构的能力。然而，在适配体设计和建模时，塑造 3D 结构的微妙原子相互作用常常被忽视，导致功能优化效率低下。确定高分辨率适体-蛋白质复合物结构仍然面临挑战。此外，通过实验确定的具有精细功能的DNA分子的3D结构仍然很少。这些因素阻碍了我们对一些重要DNA适体的理解和优化。在这里，我们对 41-nt sgc8c 进行了基于简化溶液 NMR 的结构研究，这是一种用于靶向膜蛋白酪氨酸激酶 7 的重要 DNA 适体，用于癌症治疗诊断。我们证明 sgc8c 预折叠成复杂的三向连接（3WJ）结构，通过长程三级相互作用和广泛的碱基堆叠来稳定。通过 NMR 化学位移扰动、定点突变和 3D 结构信息，我们确定了构成 sgc8c 关键功能元件的必需核苷酸，这些元件集中在 3WJ 的核心。利用完善的结构-功能关系，我们通过修改顶端环并引入L-DNA碱基对，有效地设计了两个sgc8c变体，以同时增强热稳定性、生物稳定性以及对蛋白质和细胞靶标的结合亲和力，这是以前从未实现过的壮举尽管付出了巨大的努力。这项工作展示了一种基于 NMR 的简化方法来理解和优化 sgc8c，而无需获取复杂的结构，并为 DNA 分子的复杂结构功能组织提供了原理。

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.