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

Joseph A. Gredell, Angela K. Berger, S. Patrick Walton

Research output: Contribution to journalArticle

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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, ̃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.

LanguageEnglish (US)
Pages744-755
Number of pages12
JournalBiotechnology and Bioengineering
Volume100
Issue number4
DOIs
StatePublished - Jul 1 2008

Profile

Small Interfering RNA
Messenger RNA
Gene Silencing
Genes
Hep G2 Cells
RNA Interference
RNA
HeLa Cells
Free energy
Transfection
Structural properties
Nucleation
Proteins

Keywords

  • Guide strand structure
  • MRNA structure
  • RNA interference
  • SiRNA
  • Small interfering RNA
  • Target

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Impact of target mRNA structure on siRNA silencing efficiency : A large-scale study. / Gredell, Joseph A.; Berger, Angela K.; Walton, S. Patrick.

In: Biotechnology and Bioengineering, Vol. 100, No. 4, 01.07.2008, p. 744-755.

Research output: Contribution to journalArticle

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