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Then, this hypothetical optimal binding partner was mutated stepwise as follows: A random position in the sequence was chosen to be substituted with 75% probability, deleted with 17% or a nucleotide inserted (8%).
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In standard MST one binding partner is fluorescently labeled.
On the contrary, we observed bimodality (Figure 4D) when the SigA binding site was mutated.
When the 124/506B binding site was mutated, miR-124 still upregulated lucifease expression to 240% (p = 0.0030).
When the 148/152A binding site was mutated, miR-148 downregulated reporter gene expression to 47% (p = 0.0049).
When the 124/506A binding site was mutated, miR-124 no longer affected expression of the reporter gene.
When the binding site was mutated, no effect was observed compared with control vector.
This response was abolished when the miR-29 binding site was mutated.
Moreover, the inhibition was partially rescued when one of the binding sites was mutated or almost fully relieved with all sites mutated.
The TCF4/Lef1 binding site "TTTGA" was mutated to "CCTTG" and exactly same mutations were introduced at both the binding sites.
The SOX2 HMG-binding domain recognition site was mutated with a site-directed mutagenic primer.
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