Benimetskaya Luba, Guzzo-Pernell Nancy, Liu Su-Ting, Lai Johnathan C H, Miller Paul, Stein C A
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, 3052, Australia.
Bioconjug Chem. 2002 Mar-Apr;13(2):177-87. doi: 10.1021/bc010068j.
The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.
反义技术的发展主要集中在改进寡核苷酸导入细胞的方法。在本研究中，我们描述了一种基于双功能肽的寡核苷酸递送新策略，该双功能肽由一个C端鱼精蛋白片段（包含一个DNA结合结构域）和一个源自SV40大T抗原的N端核定位信号序列组成（其中两种肽的序列分别为R6WGR6-PKKKRKV [s-鱼精蛋白-NLS]和R4SR6FGR6VWR4-PKKKRKV [l-鱼精蛋白-NLS]）。我们通过内在荧光猝灭证明，这类肽能与寡脱氧核苷酸形成复合物。为了评估递送效果，我们使用了一种靶向PKC-α mRNA 3'-非翻译区的20聚体硫代磷酸酯寡聚物（Isis 3521）和一种靶向人bcl-2开放阅读框前六个密码子的18聚体硫代磷酸酯G3139，并将它们与两种肽（s-或l-鱼精蛋白-NLS）中的任何一种复合。共聚焦显微镜显示，这些肽能结合反义寡核苷酸并将其递送至T24膀胱癌细胞和PC3前列腺癌细胞的细胞核。此外，如蛋白质免疫印迹法和Northern印迹法所示，肽-寡核苷酸复合物能显著下调互补mRNA的表达，进而导致蛋白质表达下调。然而，在某些情况下（主要是在PC3细胞中），需要用氯喹处理细胞才能产生反义活性。使用该策略，在寡聚物最低浓度为0.25 microM并结合12 - 15 microM肽的情况下，T24和PC3细胞中的PKC-α蛋白和mRNA表达以及PC3细胞中的bcl-2表达至少降低了约75±10%。基于我们的结果，我们得出结论，这类富含精氨酸的肽可能是用于细胞递送反义寡核苷酸的潜在有用载体。这种新策略可能比其他寡核苷酸递送方法具有几个优势，并且可能补充现有的基于脂质的技术。
