Designing siRNA that distinguish between genes that differ by a single nucleotide.
Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, whi...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2006-09-01
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| Series: | PLoS Genetics |
| Online Access: | http://europepmc.org/articles/PMC1560399?pdf=render |
| _version_ | 1818290833476026368 |
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| author | Dianne S Schwarz Hongliu Ding Lori Kennington Jessica T Moore Janell Schelter Julja Burchard Peter S Linsley Neil Aronin Zuoshang Xu Phillip D Zamore |
| author_facet | Dianne S Schwarz Hongliu Ding Lori Kennington Jessica T Moore Janell Schelter Julja Burchard Peter S Linsley Neil Aronin Zuoshang Xu Phillip D Zamore |
| author_sort | Dianne S Schwarz |
| collection | DOAJ |
| description | Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide. |
| first_indexed | 2024-12-13T02:34:27Z |
| format | Article |
| id | doaj.art-eb326f6c2d474e3fb8c5df12834e5b11 |
| institution | Directory Open Access Journal |
| issn | 1553-7390 1553-7404 |
| language | English |
| last_indexed | 2024-12-13T02:34:27Z |
| publishDate | 2006-09-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Genetics |
| spelling | doaj.art-eb326f6c2d474e3fb8c5df12834e5b112022-12-22T00:02:26ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042006-09-0129e14010.1371/journal.pgen.0020140Designing siRNA that distinguish between genes that differ by a single nucleotide.Dianne S SchwarzHongliu DingLori KenningtonJessica T MooreJanell SchelterJulja BurchardPeter S LinsleyNeil AroninZuoshang XuPhillip D ZamoreSmall interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.http://europepmc.org/articles/PMC1560399?pdf=render |
| spellingShingle | Dianne S Schwarz Hongliu Ding Lori Kennington Jessica T Moore Janell Schelter Julja Burchard Peter S Linsley Neil Aronin Zuoshang Xu Phillip D Zamore Designing siRNA that distinguish between genes that differ by a single nucleotide. PLoS Genetics |
| title | Designing siRNA that distinguish between genes that differ by a single nucleotide. |
| title_full | Designing siRNA that distinguish between genes that differ by a single nucleotide. |
| title_fullStr | Designing siRNA that distinguish between genes that differ by a single nucleotide. |
| title_full_unstemmed | Designing siRNA that distinguish between genes that differ by a single nucleotide. |
| title_short | Designing siRNA that distinguish between genes that differ by a single nucleotide. |
| title_sort | designing sirna that distinguish between genes that differ by a single nucleotide |
| url | http://europepmc.org/articles/PMC1560399?pdf=render |
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