PDF(Pubmed)
Abstract：
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.
摘要：
DNA适体由于其对蛋白质靶标的高结合亲和力和特异性，已成为疾病疗法中的新型分子工具。这依赖于它们折叠成独特的三维(3D)结构的能力。然而,在设计和建模适体时，通常会忽略塑造3D结构的精细原子相互作用，导致低效的功能优化。在确定高分辨率适体-蛋白质复合物结构方面存在挑战。此外，实验确定的具有精致功能的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。这里,我们对41-ntsgc8c进行了基于NMR的流线型溶液结构研究，一种突出的DNA适体，用于靶向膜蛋白酪氨酸激酶7，用于癌症治疗。我们表明，sgc8c预折叠成复杂的三通接头（3WJ）结构，该结构通过远程三级相互作用和广泛的基基堆叠而稳定。通过NMR化学位移扰动描绘，定点诱变，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，这些核苷酸集中在3WJ的核心。利用完善的结构-功能关系，我们通过修饰顶环和引入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，尽管付出了广泛的努力，但以前没有实现的壮举。这项工作展示了一种简化的基于NMR的方法来理解和优化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。
