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Decoys and Tough Decoys (TuDs)

We wanted something that would provide a tight double repression system with a very distinct change between on and off. The TuDs and Decoys design were originally inspired by the “Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells,” paper. We copied their designs and wanted to reproduce the results in our lab. To do so, we ordered TuDs and decoys both with and without bulges. The bulges are designed to disrupt RISC complex activity; something which degrades short RNA like our decoys in the cell.

TuDs and Decoys are thought to work by titrating away silencing micro RNA strands in the cell before they interact with the coding mRNA. They must be tested in a specifically designed in vivo circuit that includes the signal protein, the corresponding miRNA, and finally the Decoy which is being tested.

FF1 Knockdown

In order to transcribe microRNAs, we first needed to characterize the U6 promoter. To do so, we utilized a system that constitutively expresses eYFP with a microRNA site attached. Adding the U6:miRNA, we see knockdown of the gene. By varying molar ratios of miRNA to fluorescent signal, we can calibrate the strength of the U6 promoter. Another advantage to this system is that it can serve as an actuation method: a miRNA targeting a gene of interest could be the output to a RNA circuit.

100,000 HEK293 Cells were transfected with varying molar ratios of U6-tetO:mirFF1 to Hef1a:eYFP 4xFF1, and 1:1 molar ratio of Hef1a:eYFP 4xFF1 and Hef1a:TagBFP (a transfection marker), standardized to a total of 500ng plasmid DNA using 1.65 uL of lipofectamine 2000. As the ratio of mirFF1 increases from 0.25X to 8X, there is a corresponding decrease in the yellow fluorescent signal, indicating gene knockdown. The histograms show the population of cells shifting from yellow towards the non-fluorescent region, by 102. This region was determined by analyzing a no-transfection control.

Controlling mRNA Levels Using Hammerhead Ribozymes

Software rendering of the minimum free energy structure of the  Hammerhead ribozyme Software rendering of the minimum free energy structure of the  Hammerhead ribozyme
Software rendering of the minimum free energy structure of the Hammerhead ribozyme. Left: abstract ball-and-chain representation, right: 3D rendering of RNA.