Department of Biological Sciences, Missouri University of Science and Technology, Rolla, MO, USA.
Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien, Taiwan.
Methods Mol Biol. 2021;2211:113-121. doi: 10.1007/978-1-0716-0943-9_9.
Cell-penetrating peptides (CPPs) are small peptides which help intracellular delivery of functional macromolecules, including DNAs, RNAs, and proteins, across the cell membrane and into the cytosol, and even into the nucleus in some cases. Delivery of macromolecules can facilitate transfection, aid in gene therapy and transgenesis, and alter gene expression. L5a (RRWQW), originally derived from bovine lactoferricin, is one kind of CPPs which can promote cellular uptake of plasmid DNA and enters cells via direct membrane translocation. The peptide complexes noncovalently with DNA over a short incubation period. DNA plasmid and L5a complex stability is confirmed by a decrease in mobility in a gel retardation assay, and successful transfection is proven by the detection of a reporter gene in cells using fluorescent microscopy. Here, we describe methods to study noncovalent interactions between L5a and plasmid DNA, and the delivery of L5a/DNA complexes into cells. L5a is the one of the smallest CPPs discovered to date, providing a small delivery vehicle for macromolecules in mammalian cells. A small vehicle which can enter the nucleus is ideal for efficient gene uptake, transfer, and therapy. It is simple to complex with DNA plasmids, and its nature allows mammalian cells to be easily transfected.
细胞穿透肽（CPPs）是一类小分子肽，可帮助将包括 DNA、RNA 和蛋白质在内的功能大分子穿过细胞膜并进入细胞质，在某些情况下甚至进入细胞核。大分子的递呈可以促进转染，有助于基因治疗和转基因，以及改变基因表达。L5a（RRWQW）最初来源于牛乳铁蛋白，是一种 CPP，可以促进质粒 DNA 的细胞摄取，并通过直接膜转位进入细胞。该肽在短孵育期内与 DNA 非共价结合。通过凝胶阻滞试验证实 DNA 质粒和 L5a 复合物的稳定性降低，并且通过荧光显微镜检测细胞中的报告基因证明了成功的转染。在这里，我们描述了研究 L5a 和质粒 DNA 之间非共价相互作用以及将 L5a/DNA 复合物递送至细胞中的方法。L5a 是迄今为止发现的最小 CPP 之一，为哺乳动物细胞中的大分子提供了一种小型递药载体。能够进入细胞核的小型载体对于有效摄取、转移和治疗基因非常理想。它与 DNA 质粒的复合物很简单，其性质允许容易转染哺乳动物细胞。
