Document Detail


RNA interference from multimeric shRNAs generated by rolling circle transcription.
MedLine Citation:
PMID:  17155910     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Attila A Seyhan; Alexander V Vlassov; Brian H Johnston
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural    
Journal Detail:
Title:  Oligonucleotides     Volume:  16     ISSN:  1545-4576     ISO Abbreviation:  Oligonucleotides     Publication Date:  2006  
Date Detail:
Created Date:  2006-12-12     Completed Date:  2007-01-12     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101188415     Medline TA:  Oligonucleotides     Country:  United States    
Other Details:
Languages:  eng     Pagination:  353-63     Citation Subset:  IM    
Affiliation:
SomaGenics, Inc., Santa Cruz, CA 95060, USA.
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MeSH Terms
Descriptor/Qualifier:
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:
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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