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.

[1]  Anton P. McCaffrey,et al.  Inhibition of hepatitis B virus in mice by RNA interference , 2003, Nature Biotechnology.

[2]  B. Cullen,et al.  by RNA Interference Immunodeficiency Virus Type 1 Replication Potent and Specific Inhibition of Human , 2002 .

[3]  R. Bernards,et al.  Stable suppression of tumorigenicity by virus-mediated RNA interference. , 2002, Cancer cell.

[4]  M. Amarzguioui,et al.  Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. , 2002, Nucleic acids research.

[5]  J. Church,et al.  siRNA-DIRECTED INHIBITION OF HIV-1 INFECTION , 2003, Pediatrics.

[6]  B. Li,et al.  Expression profiling reveals off-target gene regulation by RNAi , 2003, Nature Biotechnology.

[7]  B. Ramratnam,et al.  Human Immunodeficiency Virus Type 1 Escape from RNA Interference , 2003, Journal of Virology.

[8]  Anastasia Khvorova,et al.  RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Martin Tabler,et al.  Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila , 2001, Current Biology.

[10]  M. Stevenson,et al.  Modulation of HIV-1 replication by RNA interference , 2002, Nature.

[11]  M. Amarzguioui,et al.  Tolerance for mutations and chemical modifications in a siRNA. , 2003, Nucleic acids research.

[12]  D. Engelke,et al.  Effective expression of small interfering RNA in human cells , 2002, Nature Biotechnology.

[13]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[14]  R. Medzhitov,et al.  Retroviral delivery of small interfering RNA into primary cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Ali Ehsani,et al.  Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells , 2002, Nature Biotechnology.

[16]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[17]  Sebastian Bonhoeffer,et al.  Rapid production and clearance of HIV-1 and hepatitis C virus assessed by large volume plasma apheresis , 1999, The Lancet.

[18]  K. Taira,et al.  U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells , 2002, Nature Biotechnology.

[19]  S. Reske,et al.  Gene silencing by adenovirus‐delivered siRNA , 2003, FEBS letters.

[20]  R. Andino,et al.  Short interfering RNA confers intracellular antiviral immunity in human cells , 2002, Nature.

[21]  Mamoru Watanabe,et al.  Inhibition of intracellular hepatitis C virus replication by synthetic and vector‐derived small interfering RNAs , 2003, EMBO reports.

[22]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[23]  B. Ramratnam,et al.  Promoter choice affects the potency of HIV-1 specific RNA interference. , 2003, Nucleic acids research.

[24]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.