In a study published in PNAS, a research team led by Prof. TAN Weihong, Prof. HAN Da, and Assoc. Prof. GUO Pei from the Hangzhou Institute of Medicine (HIM) of the Chinese Academy of Sciences (CAS) resolved the first high-resolution structure of the sgc8c DNA aptamer that targets protein tyrosine kinase 7 (PTK7), engineered two optimal sgc8c variants for disease theranostics efficiently, and revealed new principles for the sophisticated structural and functional organization of DNA molecules.
Aptamers are functional nucleic acids that have broad applications in clinical diagnosis and targeted drug delivery. The high binding affinity and specificity of an aptamer for its protein target depend on its intricate three-dimensional (3D) structure.
The 3D structure of an aptamer in complex with its protein partner helps to understand and optimize its functionality. However, the complex structure is difficult to be obtained due to the conformational heterogeneity of the aptamer and/or protein, and the 3D structures of DNA molecules, which are perceived to lack RNA-like tertiary interactions, remain largely unexplored.
Sgc8c is a 41-nt DNA aptamer screened through cell-SELEX to target leukemia cells. The molecular target of sgc8c is PTK7, a transmembrane receptor pseudokinase that is overexpressed in various types of cancers. Owing to its high binding affinity and specificity for both protein and cell targets, sgc8c has become one of the most widely used DNA aptamers in cancer theranostics. However, the structural basis underlying the functionality of sgc8c remains elusive, and the structure-guided functional understanding and optimization of sgc8c are needed.
In this study, the researchers first probed ten Watson–Crick base pairs in sgc8c using solution NMR and identified three paired regions including P1, P2 and P3. They then integrated the use of NMR chemical shift perturbations (CSPs) and site-directed mutagenesis assays to confirm that nucleotides from P2 constituted the key binding element.
After consolidating that binding to PTK7 did not perturb the original 3D fold of sgc8c, they determined the solution NMR structure of sgc8c and elucidated an intricate three-way junction fold stabilized by long–range hydrogen bonding and extensive base–base stacking interactions.
Several tertiary interactions, commonly observed in RNA but rarely found in DNA molecules, are crucial to maintain the structure and function of sgc8c. Most intriguingly, sgc8c can recruit more than ten nucleotides from distinct regions and assemble them into its key structural and functional framework.
Guided by the well-established structural and functional relationship, the researchers have efficiently engineered two optimal sgc8c variants that exhibit simultaneously enhanced thermostability, biostability, and binding affinity to both protein and cell targets, providing new avenues for diverse aptamer-based biomedical applications.
This work develops a streamlined NMR-based approach to overcome challenges in understanding and optimizing the function of DNA aptamers that target membrane proteins, and highlights the pivotal role of tertiary interactions in shaping the intricate structure and sophisticated function of DNA molecules.
This work was funded by the National Key R&D Program of China, and the National Natural Science Foundation of China.
Solution NMR structure of the sgc8c DNA aptamer
HIM, the first and only institution that focuses on the medicine in CAS, was established in 2019. Driven by the basic sciences and technology, HIM views molecular medicine as the key to addressing fundamental issues in tumor diagnosis, targeted treatment, novel drug R&D, and smart healthcare, as well as basic and applied research related to the diagnosis, treatment, prevention and control of major diseases. Over the last five years, HIM has made remarkable progress in solving clinical problems through interdisciplinary integration.
Contact: GUO Pei
Hangzhou Institute of Medicine of the Chinese Academy of Sciences
E-mail: guopei@ibmc.ac.cn
Reference: Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization
https://www.pnas.org/doi/10.1073/pnas.2404060121
Online time: July 11, 2024
