Kubo Takanori, Zhelev Zhivko, Ohba Hideki, Bakalova Rumiana
On-Site Sensing and Diagnosis Research Laboratory, AIST-Kyushu, Tosu, Japan.
Oligonucleotides. 2007 Winter;17(4):445-64. doi: 10.1089/oli.2007.0096.
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 approximately 100 times more stable than 21-nt siRNA and 21-nt dsRNA in cell-cultured medium supplemented with 10% inactivated serum, approximately 50 times more stable in 90% inactivated serum, and approximately 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.
本研究描述了27个核苷酸的双链RNA（dsRNAs）相较于21个核苷酸的小干扰RNA（siRNAs）具有改进的特性，并着重指出利用其修饰物和缀合物在活细胞和动物中实现直接长期基因沉默的可能性，而无需使用传统的转染剂。通过使用海肾荧光素酶基因沉默系统和培养的细胞系，我们确定27个核苷酸的dsRNAs具有比21个核苷酸的siRNAs和21个核苷酸的dsRNAs高约三到五倍的“长期”RNA干扰活性。此外，如果在将RNA双链体转染到细胞之前，先将其与细胞培养基预孵育数小时，21聚体完全失去其RNA干扰效应，而27聚体、其氨基修饰物、硫醇修饰物和胆固醇缀合物则表现出强烈的基因沉默作用。为了阐明27个核苷酸的dsRNAs具有更高RNA干扰活性的原因，我们发现它们在补充有10%灭活血清的细胞培养基中比21个核苷酸的siRNA和21个核苷酸的dsRNA稳定约100倍，在90%灭活血清中稳定约50倍，在活性血清中稳定约6倍。5'正义链修饰被选为最稳定、可被Dicer酶识别且具有最高RNA干扰潜力的修饰。5'正义链修饰的RNA干扰活性甚至高于未修饰的27个核苷酸的dsRNA。5'正义链氨基修饰也不影响21个核苷酸的siRNA、右悬端25/27个核苷酸（R25D/27）和25D/27个核苷酸RNA的活性。5'正义链修饰的R25D/27个核苷酸和25D/27个核苷酸RNA在血清中的稳定性低于平头27个核苷酸的dsRNA。然而，这些不对称RNA比修饰和未修饰的平头27个核苷酸的dsRNAs更具活性，这证明了不对称设计的优越性。5'正义链修饰被认为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RNA干扰能力，并确保在活细胞和动物中实现快速长期基因沉默的可能性。5'正义链与胆固醇的缀合证实了这一假设。
