Question

1. Describe how small interfering RNA (siRNA) can be used to inhibit gene expression.

2. How can we assess the effectiveness of siRNA at silencing our target genes?

Answer 1

Small Interfering RNA

• Small interfering RNAs (siRNAs) are artificially synthesized 19–23 nucleotide long double-stranded RNA molecules.
• They are routinely used in molecular biology for transient silencing of gene of interest. They elicit RNAi response upon binding to their target transcript based on the sequence complementarity.
• They have been rightly used to study the effect of various oncogenic lncRNAs through the loss of function. Using siRNAs, a high degree of silencing was observed against the lncRNA HOTAIR.
• A strong anticancer phenotype was observed both in vitro and in vivo.57,80Similarly, MALAT1 was targeted using siRNAs and a markedly reduced cell proliferation was observed in hepatocellular carcinoma.81 In fact, many siRNA drugs against protein coding mRNAs are already in phase I and phase II clinical trials for testing against cancer.82 For example, siRNA–EphA2–DOPC against EphA2, TKM-080301 targeting PLK1, and CALAA-01 inhibiting RRM2 are in phase I clinical trial.
• Further, siRNA drug siG12D LODER and Atu027 are being explored as anticancer drugs in combination of conventional chemotherapeutic substances.82 The performance of these siRNA drugs will pave the way for more clinical trials for targeting oncogenic/onco-promoting lncRNAs. However, there are certain unavoidable caveats for developing siRNAs as drug molecules in cancer.
• The machinery for RNAi, the mechanism behind siRNAs function, is located in the cytoplasm. Therefore, it will be difficult to target nuclear-restricted lncRNAs.
• Another obstacle for using siRNA is the lack of availability of a suitable delivery system. Most of the in vitro studies with siRNAs are conducted using a transfection agent that cannot be used for in vivo delivery.
• However, many studies are in progress for conjugating the siRNA drug with nanoparticles that seem to be an effective vehicle for carrying siRNAs.
• Small interfering RNAs (siRNAs) can specifically inhibit geneexpression. As a result, they have tremendous scientific and clinical potential. However, the use of these molecules in patients and animal models has been limited by challenges with delivery. Intracellular RNA delivery is difficult; it requires a system that protects the siRNA from degradative nucleases in the bloodstream, minimizes clearance by the reticuloendothelial system, maximizes delivery to the target tissue, and promotes entry into, and out of, an endocytic vesicle.
• Despite these barriers, recent data suggest that RNA may be targeted to cells of interest in vivo.
• This gives outline strategies for targeted siRNA delivery, and description of how these strategies may be improved.

Can be Assessed by

By quantitative RT-PCR in cell-based assays, revealing that over 60% of siRNA sequences designed by DSIR silenced their target genes by at least 70%. Silencing efficacy was sustained even when low siRNA concentrations were used.

• Assessing DSIR Design by Measuring siRNA Activity

• Relationship between siRNA Efficacy and Potential off-target Effects