Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins
Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins
复制标题
DOI：
10.1073/pnas.0807883106
复制
发表时间：
2009-04-14
影响因子：
11.1
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
McNaughton, Brian R.;Cronican, James J.;Liu, David R.
核酸试剂，包括小干扰RNA（siRNA）和质粒DNA，是研究哺乳动物细胞的重要工具，并且是开发新治疗剂的有希望的起点。然而，实现其全部潜力需要制备简单、对许多哺乳动物细胞系有效且无毒的核酸递送试剂。我们最近描述了广泛的表面诱变的蛋白质的方式，显着增加其净电荷。在这里，我们报告说，带正电的绿色荧光蛋白，包括一个理论净电荷为+36（+36 GFP）的变体，可以穿透各种哺乳动物细胞系。+36 GFP的内化依赖于与存在于大多数哺乳动物细胞表面上的硫酸化蛋白聚糖的非特异性静电相互作用。当+36 GFP与siRNA混合时，形成直径约为1.7 μ m的蛋白质-siRNA复合物。将这些复合物添加到五种哺乳动物细胞系中，包括对阳离子脂质介导的siRNA转染具有抗性的四种细胞系，导致有效的siRNA递送。在这五种细胞系中的四种中，由+36 GFP转染的siRNA抑制靶基因表达。我们表明，+36 GFP对蛋白水解具有抗性，在血清存在下是稳定的，并且延长了siRNA和与其复合的质粒DNA的血清半衰期。+36 GFP的变体可以介导DNA转染，使基于质粒的基因表达成为可能。这些发现表明，带超正电荷的蛋白质可以克服目前使用的转染剂的一些关键限制。
Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of +36 (+36 GFP), can penetrate a variety of mammalian cell lines. Internalization of +36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When +36 GFP is mixed with siRNA, protein-siRNA complexes approximate to 1.7 mu m in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by +36 GFP suppresses target gene expression. We show that +36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of +36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.