Pusch Oliver, Boden Daniel, Silbermann Rebecca, Lee Fred, Tucker Lynne, Ramratnam Bharat
Laboratory of Retrovirology, Division of Infectious Diseases, 55 Claverick Street, 4th floor, Department of Medicine, Brown Medical School, Providence, RI 02903, USA.
Nucleic Acids Res. 2003 Nov 15;31(22):6444-9. doi: 10.1093/nar/gkg876.
The degradation of a selected mRNA species by RNA interference requires a high degree of homology between the short interfering or short hairpin RNA (si or shRNA) and its target. Recent reports have demonstrated that the number and location of nucleotide mismatches affect the activity of si/shRNA. Here, we systematically examined the effect of single nucleotide mutations in all 21 positions of an effective shRNA that targets the gag gene of HIV-1. We found that all mutant shRNAs exerted RNAi activity but were less effective in gene silencing compared to the wild-type gag shRNA. The most pronounced reduction in function was observed with mutations in the central and 5' regions of the shRNA. Our results demonstrate that optimal gene silencing requires perfect homology between shRNA and the chosen target, but that a variable degree of silencing occurs, depending upon the precise location of nucleotide mismatches.
通过RNA干扰对选定的mRNA种类进行降解，需要短干扰RNA或短发夹RNA（siRNA或shRNA）与其靶标之间具有高度同源性。最近的报道表明，核苷酸错配的数量和位置会影响siRNA/shRNA的活性。在这里，我们系统地研究了针对HIV-1 gag基因的有效shRNA的所有21个位置上单核苷酸突变的影响。我们发现，所有突变的shRNA都具有RNA干扰活性，但与野生型gag shRNA相比，其基因沉默效果较差。在shRNA的中央和5'区域发生突变时，功能下降最为明显。我们的结果表明，最佳的基因沉默需要shRNA与选定靶标之间具有完美的同源性，但根据核苷酸错配的精确位置，会发生不同程度的沉默。
