NanoThera Lab, Department of Biomedical Engineering, Erciyes University, Kayseri, Turkey.
ERNAM - Nanotechnology Research and Application Center, Erciyes University, Kayseri, Turkey.
Methods Mol Biol. 2022;2434:117-128. doi: 10.1007/978-1-0716-2010-6_7.
Nanomaterials have aroused attention in the recent years for their high potential for gene delivery applications. Most of the nanoformulations used in gene delivery are positively charged to carry negatively charged oligonucleotides. However, excessive positively charged carriers are cytotoxic. Therefore, the complexed oligonucleotide/nanoparticles should be well-examined before the application. In that manner, agarose gel electrophoresis, which is a basic method utilized for separation, identification, and purification of nucleic acid molecules because of its poriferous nature, is one of the strategies to determine the most efficient complexation rate. When the electric field is applied, RNA fragments can migrate through anode due to the negatively charged phosphate backbone. Because RNA has a uniform mass/charge ratio, RNA molecules run in agarose gel proportional according to their size and molecular weight. In this chapter, the determination of complexation efficiency between cationic polymer carriers and small interfering RNA (siRNA) cargos by using agarose gel electrophoresis is described. siRNA/cationic polymer carrier complexes are placed in an electric field and the charged molecules move through the counter-charged electrodes due to the phenomenon of electrostatic attraction. Nucleic acid cargos are loaded to cationic carriers via the electrostatic interaction between positively charged amine groups (N) of the carrier and negatively charged phosphate groups (P) of RNA. The N/P ratio determines the loading efficiency of the cationic polymer carrier. In here, the determination of N/P ratio, where the most efficient complexation occurs, by exposure to the electric field with a gel retardation assay is explained.
近年来，纳米材料因其在基因传递应用方面的巨大潜力而受到关注。大多数用于基因传递的纳米制剂都是带正电的，以携带带负电的寡核苷酸。然而，过多的带正电荷的载体具有细胞毒性。因此，在应用之前，应该对复合的寡核苷酸/纳米颗粒进行充分的检查。在这种情况下，琼脂糖凝胶电泳是一种基本的方法，由于其多孔性，可用于分离、鉴定和纯化核酸分子，是确定最有效复合物形成率的策略之一。当施加电场时，由于带负电荷的磷酸骨架，RNA 片段可以通过阳极迁移。由于 RNA 具有均匀的质量/电荷比，RNA 分子在琼脂糖凝胶中根据其大小和分子量按比例运行。在本章中，描述了通过琼脂糖凝胶电泳测定阳离子聚合物载体和小干扰 RNA(siRNA)载体之间复合物形成效率的方法。siRNA/阳离子聚合物载体复合物置于电场中，带电荷的分子通过静电吸引现象穿过带相反电荷的电极移动。核酸载体通过载体上带正电荷的胺基（N）与 RNA 上带负电荷的磷酸基（P）之间的静电相互作用被加载到阳离子载体上。N/P 比决定了阳离子聚合物载体的负载效率。在这里，通过凝胶阻滞实验在电场中暴露来确定最有效的复合物形成的 N/P 比。
