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Frequently Asked Questions: MicroRNA Sponge Service


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Frequently Asked Questions: MicroRNA Sponge Service

1. What is the working principle of microRNA sponge?
2. How to design the sponge constructs?
3. What are the strategies for maximizing sponge expression?
4. Does the sponge method have limitations?
5. How to conduct the functional validation of the miRNA sponges?


1. What is the working principle of microRNA sponge?

Sponge RNAs, which are produced from transgenes within cells, contain complementary binding sites to a miRNA of interest. As with most miRNA target genes, the sponge’s binding sites are specific to the miRNA seed region, which allows them to block the whole family of related miRNAs. This transgenic method has proven to be a useful tool to probe miRNA functions in various experimental systems.

2. How to design the sponge constructs?

The typical sponge constructs contain 4 to 10 binding sites that are separated by a few nucleotides each. Increasing the number of binding sites may have diminishing marginal utility, as every site increases the probability of sponge RNA degradation. Variations in the bulged mismatches and the spacers can be introduced to reduce the risk of recombination during cloning and the risk of introducing unintended binding motifs for other regulatory factors. Another type of transgenic antisense inhibitor, TuD ‘‘tough decoy’’ (TuD) RNAs, place the miRNA binding sites in the single-stranded regions of short stem–loops, accurately presenting them for binding to miRNA complexes.

3. What are the strategies for maximizing sponge expression?

The efficacy of a miRNA sponge depends not just on the avidity of binding sites, but also on the concentration of sponge RNAs relative to the concentration of the miRNA. In order to maximize sponge expression, the strongest available promoter for the cell type of interest should be used. For transient assays, plasmid transfection can deliver the highest dose of sponge transgene. On the other hand, for viral delivery of sponges, transduction with high multiplicity of infection should be performed. Because random integration of the sponge transgene may disrupt an endogenous gene, it is advisable to generate multiple clonal lines or make polyclonal lines.

4. Does the sponge method have limitations?

Yes. The optimized sponges may still exhibit different degrees of inhibition in different contexts. Where miRNA concentration is very high, complete titration requires a very high and possibly unachievable dose of sponge RNA. In addition, in cells expressing a large pool of endogenous targets for the miRNA family of interest, there should be less free miRNA available, thus a lower dose of sponge RNA should suffice to give strong inhibition.

5. How to conduct the functional validation of the miRNA sponges?

The expression of a miRNA sponge does not necessarily lead to reduced miRNA levels, in other words, the binding of the miRNA: RISC complex to the sponge does not always induce miRNA decay. Therefore, mature miRNA levels, for example, quantified by qPCR or Northern blot, are a poor indicator of sponge function. If a validated target gene of the miRNA that is sponged is known, its expression level can be quantified by PCR. If the sponge successfully sequesters the specific miRNA: RISC complexes, the endogenous target mRNA should be stabilized. This may even lead to higher protein expression as determined by Western blotting.

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