Question

Question 2: The RNA interference (RNAi) pathway was discovered in C. elegans in 1993 and hs since rapidly towards therapeutic applications, including the first approved RNAi therapeutic in August of 2018. Use your u of RNAi and its therapeutic applications to answer the questions below. (A) Compare and contrast the short interfering RNA (siRNA) and micro-RNA (miRNA) processes. In what ways are these two RNAi pathways similar? In what ways do they differ? (8 points) (B) Suppose you work for a pharmaceutical company developing an RNAi therapeutic. What considerations would you need to make to ensure proper selection of the desired single-stranded RNA (from a double-stranded RNA duplex) as the "guide," RNA? How could you confirm the activity of your RNAi therapeutic in a cell culture model? (7 points)

Answer 1

1. Similarities between miRNA and siRNA

• Both are small non-coding RNAs
• Both are involved in gene regulation.
• Both target mRNA to produce gene silencing effect.
• Both act through RISC complex.

Differences between siRNA and miRNA

siRNA miRNA Full complementary to target mRNA partial complementary to mRNA Can target only one mRNA Can target multiple mRNAs Only Endonucleolytic cleavage of mRNA for gene silencing Can inhibit translation Degradation of mRNA Endonucleolytic cleavage of mRNA

2. Apply asymmetry rule: strand with a relatively unstable 5' end with U or A at position one should be selected as a guide strand. Optimal GC content should be ~30-60%. The siRNA to be designed should

• target specifically the gene of interest
• have no similarity with any other sequence other than the target to avoid the "off-target effects"
• not be longer than 30 nucleotides, as the longer siRNAs can result in activation of the IFN pathway and immune response pathways
• have multiple target sites on the same sequence to increase the efficacy

To confirm the activity of our siRNA in cell culture model:

1. You can take two sets of cells.
2. Transfect one set with the designed siRNA for our target gene.
3. Transfect the second set with a scrambled siRNA with no target.
4. Culture the cells for 6-24 hours.
5. Lyse the cells and perform western blot to look for the amount of protein produced over a period of time in these two sets of cells.
6. If the protein amount is reduced in our designed siRNA transfected cell line and not in the scrambled one, that means siRNA is working.