Nucleotide sequence homology requirements of HIV-1-specific short hairpin RNA.
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.
Pusch
Oliver
O
Laboratory of Retrovirology, Division of Infectious Diseases, 55 Claverick Street, 4th floor, Department of Medicine, Brown Medical School, Providence, RI 02903, USA.
Boden
Daniel
D
Silbermann
Rebecca
R
Lee
Fred
F
Tucker
Lynne
L
Ramratnam
Bharat
B
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015683:Gene Products, gag / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016655:HIV Core Protein p24 / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
D015683:Gene Products, gag; D016655:HIV Core Protein p24; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D012367:RNA, Viral