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

发布时间：2018-05-18 12:42

本文选题：细胞核 + 核定位序列　；参考：《核技术》2017年05期

【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。
[Abstract]:The key of gene therapy is how to transport genes into the nucleus and play a role. It is very important to observe the distribution of deoxyribonucleic acid DNA in the nucleus in real time for understanding the effect of gene therapy. Among them, the safety of vector, transport efficiency and so on are the key influencing factors of gene therapy. In this work, the complex structure of nuclear Localization signal nls) and DNA tetrahedron was constructed, and the efficiency of nuclear uptake of DNA tetrahedron was improved. The NLS and DNA tetrahedron are covalently connected by "click" chemical reaction, which solves the problem of poor stability caused by simple mixing. Furthermore, the efficiency of NLS sequences with different lengths and isoelectric points in joining to DNA tetrahedron was studied. It was found that the nonspecific binding of NLS29 with isoelectric point 4.84 to DNA was significantly lower than that of classical NLSs. The efficiency of connection reaction was increased from 37.3% to 72.3%. Both NLS12-DNA and NLS29-DNA could be targeted to the nucleus after high performance liquid chromatography (HPLC) high Performance Liquid Chromatography-HPLC, indicating that the length and isoelectric point of NLS remained very good activity. This study improved the problem that the nonspecific binding between positive NLS and negative DNA was easy to precipitate, which laid the foundation for clinical gene therapy.
【作者单位】：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;中国科学院大学;
【基金】：国家自然科学基金(No.11575278、No.21675167)资助~~
【分类号】：Q2-33;TB383.1

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