Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells
Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells
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DOI：
10.1038/12860
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发表时间：
1999-09-01
影响因子：
46.9
通讯作者：
Diamond, SL
中科院分区：
文献类型：
--
作者：
Subramanian, A;Ranganathan, P;Diamond, SL
非分裂细胞的脂质转染是低效的，因为大部分转染的DNA保留在核内体中，而逃逸到细胞质的DNA进入细胞核的速率很低。为了改善核输入的最后限速步骤，我们将含有异质核核糖核蛋白（hnRNP） A1的M9序列的非经典核定位信号（NLS）偶联到从SV40 t抗原共识NLS （ScT）的杂乱序列衍生的阳离子肽支架上。加入ScT以改善M9序列的DNA结合。质粒与M9-ScT偶联物的融合内皮细胞脂质转染后，转染率为83%，标记基因表达增加63倍。M9-ScT将定位的荧光质粒结合到渗透细胞的细胞核中，加入核孔阻断剂小麦胚芽凝集素可以阻止核的输入。这种基因转移方法可能导致非分裂细胞的无病毒和无脂质转染。
Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.