Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.
RNA interference (RNAi) is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA) encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely c...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3911983?pdf=render |
| _version_ | 1819262732070813696 |
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| author | Yongping Wang Jacqueline S Speier Jessica Engram-Pearl Robert B Wilson |
| author_facet | Yongping Wang Jacqueline S Speier Jessica Engram-Pearl Robert B Wilson |
| author_sort | Yongping Wang |
| collection | DOAJ |
| description | RNA interference (RNAi) is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA) encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3) dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools. |
| first_indexed | 2024-12-23T20:02:22Z |
| format | Article |
| id | doaj.art-999e479f38944216899160a3c51add30 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-23T20:02:22Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-999e479f38944216899160a3c51add302022-12-21T17:33:02ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0192e8739010.1371/journal.pone.0087390Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.Yongping WangJacqueline S SpeierJessica Engram-PearlRobert B WilsonRNA interference (RNAi) is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA) encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3) dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.http://europepmc.org/articles/PMC3911983?pdf=render |
| spellingShingle | Yongping Wang Jacqueline S Speier Jessica Engram-Pearl Robert B Wilson Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. PLoS ONE |
| title | Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. |
| title_full | Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. |
| title_fullStr | Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. |
| title_full_unstemmed | Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. |
| title_short | Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection. |
| title_sort | introduction of mismatches in a random shrna encoding library improves potency for phenotypic selection |
| url | http://europepmc.org/articles/PMC3911983?pdf=render |
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