目标序列二级结构对RNA干扰效果的影响

Secondary structure of target RNA impacts on RNA interference efficiency

目的探讨RNA二级结构对RNA干扰效果的影响,为RNA干扰靶序列的选择及设计有效siRNA提供依据。方法以K562细胞为模型,N-RAS基因mRNA为模板,分别针对二级结构茎区及二级结构环区设计4对siRNA,并分别采用RT-PCR、细胞活力检测及流式细胞检测等方法在RNA及细胞水平检测干扰效果差异。结果干扰结果表明,针对目标序列的siRNA均能对同源基因的表达产生一定程度的抑制,而针对二级结构环区目标序列的siR-NA干扰效果明显优于针对二级结构茎区的siRNA。结论RNA干扰效果与目标序列二级结构密切相关,在进行RNA干扰实验的目标序列选择时,应考虑二级结构的因素。

Objective To study the effects of the RNA secondary structure on the RNA interference efficacy and to optimize the RNAi target sequence selection. Methods N-Ras mRNA in K.562 human chronic erythro-leukemia cell line was selected as the target for RNAi experiments. Four siRNAs were designed aiming at secondary structure region and none-secondary structure region separately. The N-Ras expression change and the cell growth was tested by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）, light microscopy, MTI＇, and flow cytometry. Results Knock-down efficiency was recorded in all of the 4 experiment groups, but the degree varied a lot. The siRNAs targeting none-secondary structure region presented higher silencing efficiency than those targeting secondary structure region. Conclusion The secondary structure of mRNA closely relate to the RNA interference efficiency, which should be considered in the processing of the target sequence selection.