Modified 27-nt dsRNAs with dramatically enhanced stability in serum and long-term RNAi activity
Modified 27-nt dsRNAs with dramatically enhanced stability in serum and long-term RNAi activity
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DOI：
10.1089/oli.2007.0096
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发表时间：
2007-12-01
期刊：
影响因子：
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通讯作者：
Bakalova, Rumiana
中科院分区：
文献类型：
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作者：
Kubo, Takanori;Zhelev, Zhivko;Bakalova, Rumiana
本研究描述了27-nt dsRNA相对于21-nt siRNA的改进性质，并强调了使用它们的修饰和缀合物在活细胞和动物中直接长期基因沉默的可能性，避免了传统的转染子。使用海肾荧光素酶基因沉默系统和培养的细胞系，我们建立了27-nt dsRNA具有比21-nt siRNA和21-nt dsRNA高约3至5倍的“长期”RNAi活性。此外，如果RNA双链体在转染细胞前与细胞培养基预孵育数小时，则21-mer完全失去其RNAi作用，而27-mer、其氨基修饰、巯基修饰和胆固醇缀合物表现出强烈的基因沉默。在试图阐明27-nt dsRNA的较高RNAi活性的原因时，我们发现它们在补充有10%灭活血清的细胞培养培养基中的稳定性类似于21-nt siRNA和21-nt dsRNA的100倍，在90%灭活血清中的稳定性类似于50倍，并且在活性血清中的稳定性类似于6倍。选择5'有义修饰为最稳定、Dicer可接近且具有最高RNAi潜力的修饰。5'正义修饰的RNAi活性甚至高于未修饰的27-nt dsRNA的活性。5'有义氨基修饰也不影响21-nt siRNA、右突出端25/27-nt（R25 D/27）和25 D/27-nt RNA的活性。5'正义修饰的R25 D/27-nt和25 D/27-nt RNA在血清中的稳定性低于钝27-nt dsRNA。然而，这些不对称RNA比修饰和未修饰的钝27-nt dsRNA更有活性，这证明了不对称设计的优越性。5'有义修饰被认为是最适合与小信号分子缀合以促进RNA双链体的细胞内递送、保持其RNAi能力并确保在活细胞和动物中快速长期基因沉默的可能性。与胆固醇的5'有义缀合证实了这一假设。
The present study describes improved properties of 27-nt dsRNAs over 21-nt siRNAs, and accents on the possibility to use their modifications and conjugates for direct long-term gene silencing in viable cells and animals, avoiding conventional transfectants. Using a Renilla Luciferase gene-silencing system and cultured cell lines, we established that 27-nt dsRNAs possessed about three to five times higher "long-term" RNAi activity than 21-nt siRNAs and 21-nt dsRNAs. Moreover, if RNA duplexes were preincubated with cell-cultured medium for several hours before their transfection in cells, 21-mer completely lost its RNAi effect, while 27-mer, its amino modifications, thiol modifications, and cholesterol conjugates manifested a strong gene silencing. In attempts to clarify the reason(s) for the higher RNAi activity of 27-nt dsRNAs, we found that they were similar to 100 times more stable than 21-nt siRNA and 21-nt dsRNA in cell-cultured medium supplemented with 10% inactivated serum, similar to 50 times more stable in 90% inactivated serum, and similar to six times more stable in active serum. The 5' sense modification was selected as the most stable, accessible to Dicer, and with highest RNAi potential. The RNAi activity of 5' sense modifications was higher even than the activity of nonmodified 27-nt dsRNA. The 5' sense amino modification also did not influence the activity of 21-nt siRNA, right overhang 25/27-nt (R25D/27), and 25D/27-nt RNAs. The stability of 5' sense modified R25D/27-nt and 25D/27-nt RNAs in serum was lower than that of blunt 27-nt dsRNA. However, these asymmetric RNAs were more active than modified and nonmodified blunt 27-nt dsRNAs, which demonstrates the superiority of the asymmetric design. The 5' sense modifications were considered as most appropriate for conjugation with small signal molecules to facilitate the intracellular delivery of RNA duplex, to preserve its RNAi capacity, and to ensure a possibility for rapid long-term gene silencing in viable cells and animals. The 5' sense conjugation with cholesterol approved this assumption.