Successful RNAi experiments depend on siRNAs. The latest innovations in design, chemical modifications, and off-target effect prediction algorithms have been developed to produce the best performing siRNAs in the industry.

SiRNAs play a major role in RNA interference (RNAi) research, which relies on siRNA’s efficacy, potency and specificity. A combination of siRNA sequence design and chemical modifications can enhance all of these characteristics.

Silencer Select siRNAs are designed with a newly developed algorithm and include locked nucleic acid (LNA) chemical modifications, making them the best-performing siRNAs commercially available. This performance greatly increases the observation
of the same phenotype from experiment to experiment. LNA modifications were chosen after an intensive investigation into possible modifications, including 2′-OMe.

Phenotype experiments

Experiments were performed to assess the levels of knockdown and observed phenotypes. LNA-modified siRNAs consistently demonstrated the highest levels of knockdown and the least amount of off-target effects. This combination resulted in the best overall rate of expected phenotypes.

In addition to the nature of the modification, a strategic screening process was implemented to generate a novel placement pattern of LNA modifications. After an exhaustive review, the best combination and position of modifications was determined. This process resulted in the unique design of Silencer Select siRNAs.

Silencer Select siRNAs are more potent than first- and second-generation siRNA designs, and using high siRNA concentrations leads to increased off- target effects. The ability to use less siRNA but retain adequate knockdown levels is a common goal
of RNAi researchers.

Silencer Select siRNAs are up to 100 times more potent than competitor siRNAs, whether they are modified or unmodified. They are so potent that it is recommended that they be used at 3-5nM concentrations in easily transfected cells, for example, HeLa, which represents a 10-20-fold lower concentration range than most suppliers recommend. This potency is primarily due to the stringency used in siRNA design.

These siRNAs are designed using an improved algorithm that evaluates more than 90 different sequence and thermodynamic parameters to identify siRNAs with 28% better predictive accuracy than previous generation algorithms. This results in better, more consistent knockdown results than other siRNAs as measured in side-by-side comparisons.

Improving specificity

siRNA duplexes are made up of a passenger and guide strand. One way to improve siRNA specificity is to prevent the passenger strand of an siRNA duplex from interacting with the cellular machinery involved in RNAi. The LNA modifications
present on Silencer Select siRNAs enhance the guide strand bias.

The modifications in Silencer Select siRNAs do more because they reduce the number of non-targeted, differentially expressed genes detected by gene expression arrays by up to 90% as compared with unmodified siRNAs. This results in a dramatic reduction of off-target phenotypes as measured by multi-parametric, cell-based assays.

Design results

Along with LNA modifications to reduce off-target effects, the siRNA design itself serves to improve specificity. The design incorporates stringent analysis of sequence similarity to off-target transcripts, screens out potentially toxic siRNAs, analyses siRNAs for potential 3′ UTR miRNA seed region hits, and removes antiviral motifs. This results in observed phenotypes that are not primarily the result of toxicity.

In a phenotypic assay, 92% of Silencer Select siRNAs provided the expected phenotype, whereas other siRNAs elicited the expected phenotype in only 57-72% of siRNAs tested. All of these characteristics make Silencer Select siRNAs the best choice for RNAi experiments. WPF

Company profile

Life Technologies is a global biotechnology tools company dedicated to improving the human condition. Its systems, consumables and services enable researchers to accelerate scientific exploration, driving to discoveries and developments that improve life.