Genome-Wide High Potency siRNA

How do we achieve high potency & low off-target effect with SureSilencing siRNA?

The production of synthetic siRNA oligonucleotide strands begins at the 3' end. Due to practical considerations of coupling efficiency, and depending on the length and sequence of the target RNA, synthesis is often stopped before full-length siRNA are generated. These truncated forms of siRNA (see figures below) may result in sub-optimal performance, due to shifts in thermostability, alterations in specificity, changes to the seed region and sequence-related siRNA efficacy. Learn More

Shifts in thermostability: Instability of the guide strand 5' end is a dominant factor to determine the asymmetric recognition of siRNA in the process of RISC assembly (1). For the truncated forms of siRNA, the 5' end thermostability may be changed due to the difference of GC content on the 5' end. It may also make the thermostability of 5' end too close to the 3' one, which gives RISC more chances to use the sense strand as guide strand for RNAi. That will totally ruin the siRNA design and significantly increase the off-target effect.	Alteration in specificity: Well designed siRNA has only one target in the designated species. But, the truncated forms will have the second or more hits with full homology with other targets. Because the truncated forms of siRNA may still be used by RISC, it may give the "targeted" interference more targets than desired. Thus, significantly lower the siRNA specificity.
Changes to the seed region: Seed region has been confirmed to be highly related with the siRNA off-target effect (2, 3). Off-targeting can induce false positive signal during RNAi-based phenotypic screening. When a siRNA is designed, the seed region is also fixed. However, when the truncated forms of siRNA are generated, the seed region may be changed, which may cause non-specific knockdown of other genes, thus significantly increase off-target effect due to the change of seed region.	Changes to the sequence related siRNA efficacy: It is known that some key nucleotide positions are important for the efficacy of siRNA. For example, the A and U at positions 19 and 10 are involved in the efficiency of RNAi activity (4, 5). For the truncated forms of siRNA, the nucleotide at the key positions may be changed, which may cause the loss of siRNA efficacy.
Reference: Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities. Masatoshi Ichihara, Yoshiki Murakumo, Akio Masuda, Toru Matsuura, Naoya Asai, Mayumi Jijiwa, Maki Ishida, Jun Shinmi, Hiroshi Yatsuya, Shanlou Qiao, Masahide Takahashi, and Kinji Ohno. Nucleic Acids Res., Sep 2007; 35: e123. Experimental validation of the importance of seed complements frequency to siRNA specificity. Emily M. Anderson, Amanda Birmingham, Scott Baskerville, Angela Reynolds, Elena Maksimova, Devin Leake, Yuriy Fedorov, Jon Karpilow, and Anastasia Khvorova. RNA, May 2008; 14: 853 - 861. Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarities. Aimee L. Jackson, Julja Burchard, Janell Schelter, B. Nelson Chau, Michele Cleary, Lee Lim, and Peter S. Linsley. RNA, Jul 2006; 12: 1179 - 1187. Specific residues at every third position of siRNA shape its efficient RNAi activity. Takayuki Katoh and Tsutomu Suzuki. Nucleic Acids Research, 2007, Vol. 35, No. 4 e27 Rational siRNA design for RNA interference. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Nat Biotechnol. 2004 Mar; 22(3):326-30.

Purification of siRNA oligonucleotides by preparative high-performance liquid chromatography (HPLC) is the most common, and most effective, method for removing incompletely synthesized oligonucleotides and other contaminants. This additional quality control step provides siRNA with >97% purity. Therefore, HPLC-purified SureSilencing siRNA is ensured to provide high potency and low off-target effect for knockdown of your specific gene.

High Purity of SureSilencing siRNA. SureSilencing siRNA and siRNA from competitor X were analyzed side-by-side with 2100 Bioanalyzer. HPLC purified SureSilencing siRNA (center lane) demonstrated over 97% purity with right size, while siRNA from competitor X demonstrated multiple bands with shorter length siRNAs (right lane). The left lane is the molecular weight marker.

SureSilencing siRNA provide about 90% success rate. HeLa cells were transfected with 10nM of each siRNA targeting 24 transcription factors using SureFECT Transfection Reagent. 48 hours after transfection, total RNA was collected and real-time qPCR was performed to determine the target mRNA level. For each gene target, the first two siRNA designs were selected. About 90% of SureSilencing siRNA showed greater than 70% knockdown of target gene.

SureSilencing siRNA showed potent knockdown at very low concentration. HEK-293H cells were transfected with different concentration of siRNA for GAPDH using SureFECT Transfection Reagent. 48h after transfection, GAPDH activity level was determined. SureSilencing siRNA demonstrated matchless potency in effectively knocking down target gene even at 0.625 nM. Note: The usually recommended siRNA concentration for efficient knockdown (greater than 70% at mRNA level) is 10 ~ 30 nM.

SureSilencing siRNA showed extended period of knockdown. HEK-293H cells were co-transfected with turbo-RFP expression vector and either turbo-RFP SureSilencing siRNA or negative control siRNA. Expression of turbo-RFP in HEK-293H cells was monitored for 6 days (144 hours). The expression of turbo-RFP is significantly down with SureSilencing siRNA (lower panel) since Day 1 till Day 6 after transfection. In contrast, the expression of tubo-RFP wasn't affected with control siRNA (top panel).

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Genome-Wide siRNA