用RNase III制备siRNA库来诱发RNAi(2)

Figure 1. Silencing Gene Targets by RNase III Derived siRNA Cocktails. A 200 bp dsRNA (15 µg) for each gene of interest was digested with 2.5 U RNase III for 1 hour at 37℃. 1A. RNase III efficiently digests dsRNA . One microgram of the dsRNA before and after RNase III digestion was run on a 15% non-denaturing acrylamide gel along with a 21 bp chemically synthesized siRNA to GAPDH, which served as a size marker. The gel was stained with ethidium bromide and photographed under UV light. 1B. RNase III derived siRNA cocktails silence GAPDH, La and c-FOS. GAPDH and La siRNA cocktails were transfected into HeLa cells. The c-fos siRNA mixture was transfected into 293 cells followed by 24 hours of stimulation with 50 nM PMA. All samples were harvested at 48 hours post transfection and immunofluorescence was performed with the appropriate antibodies. Fluorescence signal was quantitated, normalized for cell number and graphed.
12-15bp 的RNase III 降解产物诱导基因沉默的效果
最常用于介导RNA i 的化学合成siRNA 通常是21bp 大小。已经有实验结果证实：用RNase III 在特定条件下降解得到的21bp 大小产物是非常有效的基因表达抑制剂。然而RNase III 完全消化产物是12―15bp 大小的片断，为了要研究这些12-15bp 大小的片断抑制基因表达的效果，一个200bp 的GAPDH 双链RNA 用RNase III 完全消化得到12-15bp 的产物并用聚丙烯酰胺胶纯化，按100nM 浓度转染Hela 细胞，另外化学合成的已知能有效抑制GAPDH 表达的siRNA 序列在同样浓度下转染Hela 细胞作为对照，相应的免疫荧光图象表明：12-15bp 的降解产物也能够抑制基因的表达，在相同浓度下两种方法的抑制效果相当。这个结果表明由RNase III 制备的更短一些的siRNA 混合库能够经过转染哺乳动物细胞抑制靶基因的表达，似乎没有必要通过改变反应条件或者借助纯化手段制备较长的siRNA 从而抑制靶基因的表达。改变转染的浓度，结果显示siRNA 的转染剂量对基因抑制效果有直接影响，RNase III 制备的siRNA 库需要保持较高的浓度从而达到最大的抑制效果，原因可能是由于：在这个siRNA s 库中既有一些有效的siRNA s 分子，也有很多无效的siRNA s 部分。

Figure 2. 12-15 bp RNase III Digestion Products Elicit Silencing. A 200 bp GAPDH dsRNA (30 µg) was digested with RNase III(30 U) for 1 hour at RT. Digestion products were run on a 15% non-denaturing acrylamide gel and the 12-15 bp products were excised, eluted, and ethanol precipitated. A sample was run on a 15% non-denaturing acrylamide gel for visualization (2A). HeLa cells were transfected with 100 nM of the 12-15 bp RNase III generated GAPDH siRNA s or a 21 bp chemically synthesized GAPDH siRNA . GAPDH protein levels were monitored by immunofluorescence 48 hours after transfection (2B) and the resulting images were quantitated (2C).
基因沉默的专一性
进一步的实验来验证这种方法制备的siRNA 库抑制靶基因表达的专一性：用RNase III 制备的针对GAPDH 的siRNA 库转染Hela 细胞，在降低GAPDH 表达的同时检测其他一系列非特异基因 (La, Ku-70, c-myc, ß-actin, and cdk-2) 的表达水平，结果没有检测到这些非特异基因的表达在转染前后的变化。结果显示在转染RNase III 制备的siRNA s 库后没有发生非特异基因沉默。 最近一篇关于RNase III 制备的siRNA s 和相关的RNA 结合蛋白的文章同样证实没有非特异基因沉默的发生 (5) 。在除了哺乳动物细胞以外的其他系统中，可以通过Dicer 酶复合物消化长片断RNA 双链，得到针对同一个靶基因多个位点的siRNA s 群，从而特异的抑制目的基因的表达。这些结果提示存在某种适当的机制来维护抑制反应的高度专一性。
结论
用RNase III 消化dsRNA 制备siRNA 库从而有效抑制目的基因表达，这是一种比较简单粗放、快速、性价比很高的方法。根据实验结果，导入这种方法制备的siRNA 库，并没有表现比用化学合成法制备特定的siRNA s 更高的毒性或者是对基因表达的非特异效应。采用RNase III 制备siRNA s 混合库可以避免化学合成siRNA 的昂贵费用，siRNA 表达载体的繁琐劳动，以及为找到一个有效的siRNA 序列所必经的漫长的筛选过程，因而成为有别于传统方法的制备siRNA 的另一种好办法，特别是对于只需要运用RNA i 技术作为遗传工具，快速得到RNA 干扰结果，而无需详细研究siRNA 具体信息的研究人员更是如此。

Figure 3. RNase III siRNA Cocktails Show Specificity for Silencing. HeLa cells were transfected with 100 nM RNase III generated siRNA s to GAPDH. Immunofluorescence analysis of GAPDH, La, c-MYC, Cdk-2, Ku-90, and ß-actin was performed 48 hours post transfection and subsequently quantitated.
References
1. Byrom MW, Cheng AM, Ford LP. (2003) Characterizing RNA i induced with siRNA cocktails generated by RNase III. Ambion TechNotes 10(1): 4-6.
2. Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, Bishop JM. (2002) Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci USA 99(15): 9942-7.
3. Calegari F, Haubensak W, Yang D, Huttner WB, and Bushholz F. (2002) Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA . Proc Natl Acad Sci USA 99: 14236-40.
4. Trotta R, Vignudilli T, Candini O, Intine RV, Pecorari L, Guerzoni C, santilli G, Byrom MW, Goldoni S, Ford LP, Caligiuri MA, Maraia RJ, Perrotti D, Calabretta B. (2003) Cancer Cell 3: (in press).