Construction of siRNA library for human functional genomics and hunting of RNAi-related genes.

RNA干扰（RNAi）技术已被证明在阐明各种生物的基因功能方面非常有用。已经发现合成的21 bp长的双链RNA（dsRNA），每个具有两个2nt长的3 '突出端，作为哺乳动物RNAi的短干扰RNA（siRNA）。21 bp长的siRNA依赖的RNAi在哺乳动物细胞中的作用机制已被广泛研究，我们已经建立了设计高效siRNA序列的规则。我们的指南指出，同时满足以下所有四个序列条件的21 bp长的siRNA能够在哺乳动物细胞中诱导高效的基因沉默：引导链（GS）5 '端的A/U;过客链（PS）5'端的G/C;在GS的5 '端三分之一处至少有四个A/U残基，并且不存在任何长度超过9 nt的GC延伸。还已知RNAi通过转染编码短发夹RNA（shRNA）的DNA来诱导。功能丧失的大规模筛选 ...更多信息 如果合适的编码shRNA的DNA文库可用，则突变体是可能的。为了构建编码shRNA的DNA文库，RNA聚合酶III启动子驱动的载体已被广泛使用。但是，这种pol-III驱动的系统对shRNA序列施加了各种限制，使得相当大一部分siRNA序列变得不可用。为了克服这个困难，我们开发了一种新的载体系统，它是由RNA聚合酶II启动子驱动的。现在可以设计任何有效的shRNA编码DNA，而不受任何序列限制。此外，我们发现不仅21 bp siRNA而且22 bp dsRNA是Dicer酶切的最终产物，并且显示出22 bp dsRNA的一部分作为有效的siRNA。高效22 bp siRNA的序列偏好规则与21 bp siRNA的序列偏好规则非常相似，如果不相同的话。我们还发现siRNA二聚体和三聚体能够有效地诱导RNAi，当这些寡聚体具有两个2nt长的3 '突出端并且含有关于Dicer消化的框内活性单体单元时。因此，在一些合适的细胞系中，双重或三重敲低是可能的。最后，我们使用我们的siRNA文库进行了基因筛选实验，并确定了许多人类或小鼠转录因子基因、乳腺癌相关基因和RNAi相关基因的候选基因。少

RNA interference (RNAi) has been shown quite useful in the clarification of gene function in various organisms. Synthetic 21bp long double-stranded RNAs (dsRNAs) each with two 2nt long 3'overhangs have been found to serve as short interfering RNAs (siRNAs) for mammalian RNAi. The mechanisms of 21bp-long siRNA dependent RNAi in mammalian cells have been studied extensively, and we have established the rules for designing sequences of highly effective siRNAs. Our guidelines indicate 21bp long siRNAs simultaneously satisfying all four of the following sequence conditions to be capable of inducing highly effective gene-silencing in mammalian cells : A/U at the 5'end of the guide strand (GS) ; G/C at the 5'end of the passenger strand (PS) ; at least four A/U residues in the 5'terminal third of GS and the absence of any GC stretch of more than 9nt in length. RNAi is also known to be induced by the transfection of DNA encoding short hairpin RNA (shRNA). Large scale screening of loss-of-functi … More on mutants is possible if suitable shRNA-encoding DNA libraries are available. For construction of an shRNA-encoding DNA library, RNA-polymerase-III-promoter driven vectors have been widely used. But, this pol-III-driven system imposes various restrictions on shRNA sequences so that a considerable fraction of siRNA sequences becomes unavailable. To surmount this difficulty, we developed a new vector system which is driven by RNA polymerase II promoter. It is now possible to design any effective shRNA-encoding DNA without any sequence restrictions. I addition, we found that not only 21bp siRNA but also 22bp dsRNA is the final Dicer digestion product and showed some fraction of 22bp dsRNA to serve as an effective siRNA. Sequence preference rules for highly effective 22bp siRNA were very similar, if not identical, to those for 21bp siRNAs. We also found siRNA dimer and trimer to be capable of efficiently inducing RNAi when these oligomers possess two 2nt-long 3'overhangs and contain an active monomer unit in frame with respect to Dicer digestion. Thus, double- or triple-knockdown is possible in some suitable cell lines. Finally, we carried out gene screening experiments using our siRNA libraries and identified many candidates for human or mouse transcription-factor genes, apoptosis-related genes, and RNAi-related genes. Less