Laboratory of Molecular Cell Biology, Department of Life Science, Yasuda Women's University Faculty of Pharmacy, Hiroshima, Japan.
Bioconjug Chem. 2012 Feb 15;23(2):164-73. doi: 10.1021/bc200333w. Epub 2012 Jan 26.
The development of Dicer-substrate small interfering RNAs (DsiRNAs) has been pursued in recent years because these molecules exhibit a much more potent gene-silencing effect than 21-nucleotide (nt) siRNAs. In the present study, we designed eight different types of amino-modified DsiRNAs and a palmitic acid-conjugated DsiRNA expected to result in improved biological properties of siRNAs, including their stability against nuclease degradation, membrane permeability, and RNAi efficacy. The DsiRNAs were modified with an amine at the 5'- and/or 3'-end of the sense and/or antisense strand. Dicer enzyme cleaved most of the amino-modified DsiRNAs to lead to the release of 21-nt siRNA; some of them, however, were not or partly cleaved. All amino-modified DsiRNAs exhibited strong resistance against nuclease degradations. Among the amino-modified DsiRNAs, the DsiRNA modified with an amine restricted at the 3'-end of the sense strand showed the most enhanced gene-silencing effect and maintained its potent gene suppression after one week of cell transfection against Renilla luciferase activity. For further improvement, palmitic acid was conjugated to DsiRNA at the 3'-end of the sense strand (C16-DsiRNA) to facilitate the membrane permeability and potent gene-silencing activity. The C16-DsiRNA showed enhanced membrane permeability to HeLa cells. The C16-DsiRNA exhibited extremely high inhibition of Renilla luciferase activity.
近年来，人们一直在开发 Dicer 底物小干扰 RNA（DsiRNA），因为与 21 个核苷酸（nt）siRNA 相比，这些分子表现出更强的基因沉默效果。在本研究中，我们设计了八种不同类型的氨基修饰 DsiRNA 和一种预期会改善 siRNA 生物学特性的棕榈酸偶联 DsiRNA，包括它们对核酸酶降解、膜通透性和 RNAi 功效的稳定性。DsiRNA 在正义链和/或反义链的 5'-和/或 3'-末端被修饰为胺。Dicer 酶切割大多数氨基修饰的 DsiRNA，导致释放 21-nt siRNA；然而，其中一些没有被切割或部分被切割。所有氨基修饰的 DsiRNA 都表现出很强的抗核酸酶降解能力。在氨基修饰的 DsiRNA 中，在正义链 3'-末端受限的 DsiRNA 表现出最强的基因沉默效果，并在细胞转染一周后仍能保持对 Renilla 荧光素酶活性的强基因抑制作用。为了进一步提高效果，棕榈酸被偶联到 DsiRNA 的正义链 3'-末端（C16-DsiRNA）以促进膜通透性和有效的基因沉默活性。C16-DsiRNA 对 HeLa 细胞具有增强的膜通透性。C16-DsiRNA 对 Renilla 荧光素酶活性具有极高的抑制作用。
