Endoh Tamaki, Sisido Masahiko, Ohtsuki Takashi
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
Bioconjug Chem. 2008 May;19(5):1017-24. doi: 10.1021/bc800020n. Epub 2008 Apr 29.
HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5'-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.
HIV-1反式激活因子（TAT）肽是一种细胞穿透肽（CPP），它与U1A RNA结合结构域（TatU1A）融合，以生成一种用于哺乳动物细胞的序列特异性小干扰RNA（siRNA）递送系统。该siRNA含有一个短的5'端延伸序列，可被U1A RNA结合结构域（U1AsiRNA）特异性识别。使用凝胶迁移率变动分析证实了TatU1A与U1AsiRNA的特异性结合。仅当U1AsiRNA在添加到细胞之前与TatU1A预孵育时，细胞才会将其内化。尽管大多数内化的siRNA似乎被困在内吞小室中，但通过对附着在TatU1A上的荧光团进行光刺激，可实现被困siRNA的有效重新分布。一旦进入细胞质，siRNA就会诱导RNA干扰（RNAi）介导的基因沉默。我们将这种递送策略称为CLIP-RNAi。CLIP-RNAi是RNAi实验和精准RNAi治疗的一种有前景的策略。
