| RNA interference from multimeric shRNAs generated by rolling circle transcription. | |
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MedLine Citation:
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PMID: 17155910 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Methods most commonly used for producing small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) are chemical synthesis and intracellular expression from engineered vectors. For shRNAs, chemical synthesis is very costly and construction of vectors is laborious. Synthesis by phage RNA polymerases from their natural promoters results in a 5 -terminal triphosphate that can trigger an interferon (IFN) response. Moreover, due to the requirement of phage promoters for 5 - GPuPuPu sequences for transcription initiation, shRNA transcripts may have extra 5 -nucleotides that can constrain the sequences that can be targeted. Also, the 3 ends may have an additional n + 1 nucleotide not encoded by the template. Here we present a novel approach for synthesizing functional shRNAs via rolling circle transcription (RCT) of small (approximately 70 nt) single-stranded DNA circles using T7 RNA polymerase, which avoids these issues. Due to internal pairing, these circles are dumbbell-shaped. RCT produces large transcripts (>10 kb in length) consisting of multimers (>150 copies) of shRNAs in the absence of promoter, terminator, or primer sequences. Dumbbells targeting red fluorescent protein (DsRed), human tumor necrosis factor-alpha (TNF-alpha) and hepatitis C virus (HCV) internal ribosome entry site (IRES) were prepared and transcribed. The resulting long transcripts are substrates for Dicer. When introduced into 293FT and Huh7 cells, the multimeric transcripts inhibited their target genes at levels similar to an equivalent mass of monomeric shRNAs, indicating that they can enter the RNAi pathway. Thus, rolling circle transcription of small DNA dumbbells provides a new source of biologically active interfering RNA. |
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Authors:
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Attila A Seyhan; Alexander V Vlassov; Brian H Johnston |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural |
Journal Detail:
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Title: Oligonucleotides Volume: 16 ISSN: 1545-4576 ISO Abbreviation: Oligonucleotides Publication Date: 2006 |
Date Detail:
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Created Date: 2006-12-12 Completed Date: 2007-01-12 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101188415 Medline TA: Oligonucleotides Country: United States |
Other Details:
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Languages: eng Pagination: 353-63 Citation Subset: IM |
Affiliation:
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SomaGenics, Inc., Santa Cruz, CA 95060, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Base Sequence Cell Line DNA, Circular / chemistry, genetics Hepacivirus / genetics Humans Luminescent Proteins / antagonists & inhibitors, genetics Nucleic Acid Conformation RNA Interference* RNA, Catalytic / chemistry, genetics*, metabolism* Ribonuclease III Transcription, Genetic Tumor Necrosis Factor-alpha / antagonists & inhibitors, genetics |
| Chemical | |
Reg. No./Substance:
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0/DNA, Circular; 0/Luminescent Proteins; 0/RNA, Catalytic; 0/Tumor Necrosis Factor-alpha; 0/hairpin ribozyme; 0/red fluorescent protein; EC 3.1.26.3/Ribonuclease III |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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