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PMID: 18306428 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

Biotechnology and bioengineering ·Vol. 100 ·No. 4 ·2008-07-01 ·Pages 744-55

Gredell JA, Berger AK, Walton SP

Abstract

The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate siRNAs.

MeSH Terms
Base Pairing Databases, Genetic Genetic Engineering/methods Green Fluorescent Proteins/analysis HeLa Cells Humans Models, Genetic Molecular Sequence Data RNA Interference RNA, Messenger/antagonists & inhibitors,metabolism,ultrastructure RNA, Small Interfering/pharmacology,ultrastructure Sequence Analysis, RNA Thermodynamics Transcription, Genetic/drug effects
Chemicals
RNA, Messenger RNA, Small Interfering enhanced green fluorescent protein Green Fluorescent Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gredell Joseph A
Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824-1226, USA.
Berger Angela K
Walton S Patrick
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Article Info
Journal
Biotechnology and bioengineering
Abbr.
Biotechnol Bioeng
ISSN
1097-0290
DOI
10.1002/bit.21798
Published
2008-07-01
Pages
744-55
Language
English
Region
United States
NLM ID
7502021
PMCID
PMC2658823
Subset
IM
Grants
NIGMS NIH HHS · GM079688 · United States
NIGMS NIH HHS · R01 GM079688 · United States
NIGMS NIH HHS · R01 GM079688-01 · United States
NCI NIH HHS · CA126136 · United States
NCI NIH HHS · R21 CA126136-02 · United States
NCI NIH HHS · R21 CA126136 · United States
External Links
PubMed source DOI: 10.1002/bit.21798 PMC Full Text View journal details
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