8-氧代鸟嘌呤对于端粒G-四联体DNA构象影响的纳米孔道单分子检测

The repeat unit 5'-(TTAGGG) -3'in human telomere sequence can form G-quadruplex (G4) conformation, which inhibits the activity of telomerase and promotes the apoptosis of cancer cells, and it has become a research hotspot in the treatment of cancer. However, in the G-rich telomere sequence, guanine (G) is easy to be oxidized to 8-oxoguanine (OG). Because OG cannot be effectively removed through the repair pathways, it accumulates in the telomere and affects the stability of G4 conformation. However, there is no analytical method to accurately detect the effect of OG on the G4 conformation, which greatly restricts the basic theory and application of G4 conformation as a target for cancer treatment. In order to solve this problem, the γ-hemolysin (γ-HL) nanopore with a unique internal structure is applied to accurately detect various G4 conformations at the single-molecule level. By systematically studying the current signature of OG at 12 possible positions in a single G4 conformation, the influence of the characteristic sequence of OG on the conformation and stability of G4 is investigated. Furthermore, the current signatures the human telomere containing OG modified bases was detected by the γ-HL nanopore, and the conformational information and the sequence information of OG are resolved at the single-molecular resolution.

人类端粒序列中含有的重复单元5’- (TTAGGG)-3’可形成G-四联体(G4)构象，能抑制端粒酶活性，促进癌细胞的凋亡，成为治疗肿瘤的研究热点。然而在富含G的端粒序列中，鸟嘌呤(G)易氧化成8-氧代鸟嘌呤(OG)，并且由于OG不能通过修复途径有效地去除，从而致使其在端粒中积累，对G4稳定性造成影响。然而目前尚无可以精确检测OG修饰碱基对于端粒G4构象影响的分析方法，极大的制约了将G4构象作为癌症治疗靶点的基础理论和应用研究。针对这一问题，拟利用γ-Hemolysin(γ-HL)纳米孔道的独特内部结构在单分子水平上实现多种G4构象的精准检测，通过系统研究OG位于单个G4构象的短链DNA序列12个不同位置时的电分析信号，分析OG特征序列对G4构象及其稳定性的影响。进而检测含有OG修饰碱基的人类端粒多种构象在γ-HL纳米孔道中的电分析信号，以在单分子水平上获取每种构象信息以及OG的序列信息。

作为人类遗传物质的DNA根据其序列以及相互作用的生物分子可以形成多种构象，如规范的B-型双螺旋、5mC存在时的Z-型双螺旋、DNA-RNA构成的A-型双螺旋，以及人类端粒序列中含有的重复单元5’- (TTAGGG)-3’形成G-四联体(G4)构象，均是单分子检测和生物医药靶点的研究前沿。此外，细胞内DNA存在内源性和外源性碱基损伤，进而导致其构象稳定性的变化，甚至产生新的构象。然而目前尚无可以精确检测碱基损伤对于DNA构象影响的分析方法，极大的制约了将核酸构象作为癌症治疗靶点的基础理论和应用研究。针对这一问题，本项目利用γ-Hemolysin(γ-HL)纳米孔道的独特内部结构在单分子水平上实现多种核酸构象的精准检测，精度高达90%，同时引入分子动力学对核酸在纳米孔道内折叠-去折叠动态平衡进行模拟，实现复杂电分析信号的精准解读，进而探究碱基损伤对核酸构象及其稳定性的影响。