基于核定位序列-DNA四面体复合纳米结构的细胞核成像研究  

The nuclei imaging of nucleus localization signal-DNA tetrahedron composite nanostructures

作　　者：赵彦[1,2] 李凡[1,2] 郭琳洁[1] 代江兵 邢淑[1,2] 王丽华[1] 

机构地区：[1]中国科学院上海应用物理研究所,物理生物学研究室,中国科学院界面物理与技术重点实验室,嘉定园区,上海201800 [2]中国科学院大学,北京100049

出　　处：《核技术》2017年第5期44-49,共6页Nuclear Techniques

基　　金：国家自然科学基金(No.11575278、No.21675167)资助~~

摘　　要：基因治疗的关键是如何高效转运基因进入细胞核并发挥作用,实时观察脱氧核糖核酸(Deoxyribonucleic acid,DNA)在细胞核中的分布对于了解基因治疗的效果非常重要。其中,载体的安全性、运输效率等是基因治疗的关键影响因素。本工作构建了核定位序列(Nuclear Localization Signal,NLS)与DNA四面体的复合结构,提高了细胞核摄取DNA四面体的效率。利用"点击"化学反应将NLS与DNA四面体进行共价连接,解决了简单混合带来的稳定性差等问题。进一步研究了不同长度和等电点的NLS序列在与DNA四面体连接时的效率,发现与经典的NLS_(12)相比,等电点为4.84的NLS29与DNA的非特异性结合显著降低,连接反应的效率从37.3%提高到72.3%。经高效液相色谱(High Performance Liquid Chromatography,HPLC)分离纯化后的NLS12-DNA和NLS29-DNA均可以靶向细胞核,说明改变NLS的长度和等电点后仍保持了很好的活性。该研究改善了正电NLS与负电DNA之间由于非特异性结合容易形成沉淀的问题,为临床基因治疗奠定了基础。Background： Efficient translocation of nucleic acid drugs into nucleus is a key factor in gene therapy. Therefore, real-time observation of the distribution of deoxyribonucleic acid （DNA） in the nucleus is very important to understand the effect of gene therapy. Beforehand, choosing a safe and efficient carrier for translocating DNA into nucleus is critical. Purpose： This work establishes composite structure of nucleus localization signal （NLS） and DNA tetrahedron, which improving the uptake efficiency of DNA tetrahedron into nucleus. Methods： Covalently connection NLS and DNA tetrahedron with ＂click＂ chemistry solved the problems of poor stability after simple mixing. And we further studied the connecting efficiency of different length and isoelectric point NLS with ssDNA.Results： Compared with classical NLSI2, NLS29 （of which isoelectric point is 4.84） had a low nonspecific binding. The binding efficiency of NLS29 was improved to 72.3% from 37.3%. After purification, both NLS12-DNA and NLSE9-DNA could target to cell nucleus, thus verifying that they all possessed good activity. Conclusion： This research improved nonspecific binding between positively charged NLS and negatively charged DNA, which laid a foundation for clinical gene therapy.

关 键 词：细胞核 核定位序列 DNA四面体 “点击”化学 

分 类 号：TL99[核科学技术—核技术及应用]