siRNA function in RNAi: A chemical modification analysis
siRNA function in RNAi: A chemical modification analysis
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作者：
Chiu, YL;Rana, TM
在短干扰RNA （sirna）中产生了各种化学修饰，以确定RNA干扰（RNAi）所需的生化特性。值得注意的是，sirna中戊糖2'-位置的修饰表明RNAi不需要2'-OH，这表明RNAi机制不需要2'-OH来识别sirna， RNA诱导沉默复合物（RISCs）的催化核糖核酸酶活性不涉及引导反义RNA的2'-OH。此外，与野生型siRNA相比，预测稳定siRNA的2'修饰增加了RNAi的持久性。RNAi也被化学修饰诱导，稳定了A-U碱基对之间的相互作用，表明这些类型的修饰可以提高等位基因特异性RNAi的mRNA靶向效率。改变反义siRNA-mRNA双链a型主槽结构的修饰可消除RNAi，这表明激活的RISC*需要这些双链的主槽进行识别。化学修饰单链反义RNA和双链siRNA的稳定性和RNAi活性的比较分析表明，除了siRNA的稳定性外，还有一些催化机制与RNAi效率有关。根据反义链的定义，在siRNA双链的5‘或3’部分的内部位置加入修饰或错配的核糖核苷酸表明，siRNA结构的5‘部分而不是3’部分的完整性对RNAi很重要，这突出了siRNA识别在解绕起始时的不对称性。综上所述，本研究明确了RNAi在人类细胞中的作用机制，并为RNAi介导的基因沉默应用设计有效、稳定的sirna提供了新的规则。
Various chemical modifications were created in short-interfering RNAs (siRNAs) to determine the biochemical properties required for RNA interference (RNAi). Remarkably, modifications at the 2'-position of pentose sugars in siRNAs showed the 2'-OHs were not required for RNAi, indicating that RNAi machinery does not require the 2'-OH for recognition of siRNAs and catalytic ribonuclease activity of RNA-induced silencing complexes (RISCs) does not involve the 2'-OH of guide antisense RNA. In addition, 2' modifications predicted to stabilize siRNA increased the persistence of RNAi as compared with wild-type siRNAs. RNAi was also induced with chemical modifications that stabilized interactions between A-U base pairs, demonstrating that these types of modifications may enhance mRNA targeting efficiency in allele-specific RNAi. Modifications altering the structure of the A-form major groove of antisense siRNA-mRNA duplexes abolished RNAi, suggesting that the major groove of these duplexes was required for recognition by activated RISC*. Comparative analysis of the stability and RNAi activities of chemically modified single-stranded antisense RNA and duplex siRNA suggested that some catalytic mechanism(s) other than siRNA stability were linked to RNAi efficiency. Modified or mismatched ribonucleotides incorporated at internal positions in the 5' or 3' half of the siRNA duplex, as defined by the antisense strand, indicated that the integrity of the 5' and not the 3' half of the siRNA structure was important for RNAi, highlighting the asymmetric nature of siRNA recognition for initiation of unwinding. Collectively, this study defines the mechanisms of RNAi in human cells and provides new rules for designing effective and stable siRNAs for RNAi-mediated gene-silencing applications.