应用强磁场装置研究肽核酸PNA与端粒DNA形成的G四联体结构及其对端粒酶的抑制机理

Telomerase is an enzyme which rebuilds telomeres and prevents them from shortening during cell division. Telomerase activity is completely repressed in most somatic cells and is down-regulated in stem cells during periods of cell proliferation. In contrast, over expression/activation of telomerase has been found in a broad range of human cancers (approximately 90% types). The over-activation of telomerase is necessary for the unlimited proliferation of cancer cells, which leads to immortality. Since the discovery of the pathological role of telomerase, blocking of its activity has been one of the most promising approaches to develop anti-cancer therapies. In addition, the selective activation of telomerase exclusively in cancer cells rather than normal somatic cells provides an attractive therapeutic window. As a reverse transcriptase, telomerase consists of two essential components: RNA template which binds to the single stranded guanine-rich 3′-overhang of telomeric DNA as substrate, and the catalytic subunit which adds the specific telomeric DNA sequence to the chromosome ends. Obviously the telomerase inhibition could be directly achieved through using guanine-rich PNA to target the complementary RNA template of telomerase. Nevertheless, it is also possible that this guanine-rich PNA hybridizes with guanine-rich 3′-overhang of telomeric DNA to form a stable heterodimeric G-quadruplex which is unable to be recognized by telomerase, thus indirectly inhibits telomerase. To prove this hypothesis, NMR associated technologies will be applied to investigate whether the antisense PNA has synergistic effect to inhibit telomerase via the formation of a heterodimeric G-quadruplex between PNA and telomeric DNA. Also, based on the NMR 3D structural analysis, next generation PNA probes will be developed through chemical optimization for the G-quadruplex formation, and evaluated in the in vitro anti-cancer study models. The high magnetic filed of laboratory(HMFL)has state of art magnetic related instruments such as NMR and MRI, as well as structural biology research platform, cell biology platform and drug discovery platform. This project perfectly fits the background of the collaborative grant between CAS and NSFC. We propose to integrate the structural biology, molecular biology and drug discovery efforts together by starting with interpreting the structural and functional mechanisms of the PNA/DNA Quadruplex on the basis of HMFL.

端粒酶在绝大多数肿瘤细胞中均表现出特异性过度活化，而正常体细胞中保持沉默状态，这种天然选择性使其成为具有较宽治疗窗口的抗癌药物靶标。通常认为富含鸟嘌呤G的肽核酸PNA反义核苷酸直接以端粒酶的RNA模板区为靶点实施对其的抑制作用，然而最近有证据显示它还可能同时通过与端粒富含G的DNA单链交合形成分子间G四联体结构，从而间接起到抑制端粒酶的作用，但是其具体的结构性机理还不清楚。 本项目拟依托中国科学院强磁场科学中心的强大结构生物学平台和药学平台，以结构生物学和化学生物学相结合方法，以结构药学为手段，以高频核磁和低温探头为技术支持，解析PNA－DNA复合物G四联体的三维结构，并从机理上探讨肽核酸PNA反义核苷酸探针对于端粒酶是否具有以上所述的双重抑制作用。从而为建立以核磁为基础的理论设计和开发针对端粒G四联体有高选择性靶向抗癌症药物的筛选和测试体系提供理论指导及有效性验证。

项目围绕染色体端粒区与肿瘤密切关联的关键科学和技术问题，首先通过自行设计富含鸟嘌呤G串联的核酸类似物肽核酸PNA短链探针，运用NMR和其他生化手段观测到以上这些探针能够结合端粒DNA而形成分子间非对称G四联体复合物，阐明其具有直接和间接双重抑制端粒酶的作用，实现了预期的主要研究目标。但由于PNA自身化学结构的特殊性而导致存在多重构象，造成PNA与端粒DNA之间形成的G四联体复合物NMR谱图过于复杂，不太适合下一步直接解析该复合物的原子分辨率溶液结构。经改进，项目后期尝试应用锁核酸LNA探针替代PNA后，得到了高质量NMR谱图而变得十分适合解析复合物溶液结构；特别是还发现了该LNA探针能够可控地特异性识别端粒DNA或非编码的端粒RNA(TERRA)，分别形成各自的分子间混杂型G-四联体，这就又进一步提升了LNA锁核酸探针的靶向特异性能，拓展了核酸类似物探针精确抗癌用途。目前已经收集了绝大部分实验数据，进入后期扫尾工作，并已开始论文的整理发表工作。.此外，非编码的端粒RNA（TERRA）新近被报导和端粒长度的调控密切相关，但具体机理并不明确。我们通过NMR进一步明确了端粒DNA与TERRA还能够形成DNA-RNA分子间混杂型的G-四联体复合物，应是首个原子分辨率水平上的混杂型端粒G-四联体结构，该结果可以解释端粒DNA的3’末端单链如何被TERRA保护起来，而避免细胞自身误识其为DNA损伤引发凋亡；另外也补充、拓展与端粒功能密切相关的t-loop构型如何形成的新机制；同时阐明了分别特异性靶向端粒DNA和RNA的重要性。该篇论文整理发表工作也正在准备当中。.过去针对G-四联体为靶点进行抗肿瘤老一代化合物G4小分子配体的筛选以及结构设计均是平面芳香大共轭体系，但是G4化合物仅通过π-π堆积模式而无法选择性地识别端粒DNA或端粒RNA，结果导致了抗癌治疗效果不理想。我们利用核酸类似物锁核酸LNA特殊化学结构，设计新类型的锁核酸LNA探针而实现分别特异性识别端粒DNA和RNA的目标。在核酸探针设计概念、制备方法、化学结构，端粒酶抑制机理和端粒保护新机制探索等方面取得了创新性研究成果，为新型探针的研究和发展提供了新的思路和技术支撑，为最终达到更加特异性检测、治疗癌细胞打下基础。