Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.
Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein.
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DOI：
10.1038/nbt.1541
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发表时间：
2009-06
影响因子：
46.9
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中科院分区：
文献类型：
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短干扰RNA（siRNA）诱导的RNA干扰（RNAi）反应允许进行发现研究和进行大规模筛选;然而，由于它们的大小和阴离子电荷，siRNA没有进入细胞的生物利用度。目前的方法不能以非细胞毒性方式将siRNA递送到高百分比的原代细胞中。在这里，我们报告了一种有效的siRNA递送方法，利用肽转导结构域-dsRNA结合结构域（PTD-DRBD）融合蛋白。DRBDs以高亲合力结合siRNA，掩蔽siRNA负电荷并允许PTD介导的细胞摄取。PTD-DRBD递送的siRNA以非细胞毒性的方式在原代和转化细胞的整个细胞群体中诱导快速RNAi应答，包括T细胞、HUVEC和hESC。全基因组微阵列分析显示PTD-DRBD的转录变化最小，并且我们在PBMC中未检测到任何先天免疫应答。因此，PTD-DRBD介导的siRNA递送允许对困难的原代细胞类型进行有效的RNAi操作。
Short interfering RNA (siRNA) induced RNA interference (RNAi) responses allow for discovery research and performing large scale screening; however, due to their size and anionic charge, siRNAs have no bioavailability to enter cells. Current approaches fail to deliver siRNAs into a high percentage of primary cells in a non-cytotoxic fashion. Here we report an efficient siRNA delivery approach that utilizes a Peptide Transduction Domain-dsRNA Binding Domain (PTD-DRBD) fusion protein. DRBDs bind to siRNAs with high avidity, masking the siRNA negative charge and allow for PTD-mediated cellular uptake. PTD-DRBD delivered siRNAs induced rapid RNAi responses in a non-cytotoxic manner in the entire cell population of primary and transformed cells, including T cells, HUVECs and hESCs. Whole genome microarray analysis showed minimal transcriptional changes by PTD-DRBD and we did not detect any innate immune responses in PBMCs. Thus, PTD-DRBD mediated siRNA delivery allows efficient RNAi manipulation of difficult primary cell types.