Analysis of siRNA specificity on targets with double-nucleotide mismatches.

Nucleic Acids Research
Cecilia DahlgrenZicai Liang

Abstract

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that approximately 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.

References

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Citations

Jun 16, 2011·Molecular Therapy : the Journal of the American Society of Gene Therapy·Pooja DuaDong-ki Lee
Nov 26, 2008·Oligonucleotides·Mark A Behlke
Jun 10, 2010·Retrovirology·Nick C T SchopmanBen Berkhout
Apr 3, 2010·Silence·Barbara RobertsonAnnaleen Vermeulen
Apr 23, 2010·RNA·Katrin TiemannPål Saetrom
Mar 10, 2009·PloS One·Angela LaiGreg M Arndt
Aug 20, 2011·PloS One·Firoz Ahmed, Gajendra P S Raghava
Nov 13, 2012·PloS One·Na WeiZicai Liang
Aug 23, 2008·Bioinformatics·Wuming GongTongbin Li
Jul 1, 2010·Drug Discovery Today. Technologies
Apr 25, 2009·Advanced Drug Delivery Reviews·Donald D RaoJohn Nemunaitis
Sep 15, 2011·Journal of Viral Hepatitis·X TengH-X Gu
Jan 21, 2014·Trends in Microbiology·Tim KünneStan J J Brouns
Jul 24, 2012·New Biotechnology·Joachim W Engels
Dec 6, 2018·Nature Communications·Lidiya LisitskayaAndrey Kulbachinskiy
Dec 17, 2009·Current Protocols in Nucleic Acid Chemistry·Glen F DeleaveyMasad J Damha
Apr 2, 2010·ACS Applied Materials & Interfaces·Ishtiaq SaaemJingdong Tian

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Methods Mentioned

BETA
gene knockdown
PCR
transfection
transfections
Assay

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