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.
Sources: Haraguchi et al. 2009, Kitamura 1998.

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.

The eYFP is constituitvely on, so the cell defaults to flouresing in yellow. To test the miRNA, we would induce prodution of mKate protein with eYFP specific miRNA on the introns. When the mKate is produced, the miRNA is spliced out and silences the eYFP. When the Decoys are included in the circuit, they titrate off the eYFP specific miRNA and allow the yellow floresense to continue.

FF1 Knockdown

The objective of actuation is to transduce the RNA signal output into a desired protein signal. The protein signal can range from a fluorescent protein for detection, to a protein that can induce apoptosis in the case of cancer cells. In our first step to achieve this aim, the team tested the FF1 knockdown system, as illustrated below:

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.

In this circuit, HEK293 cells transfected with pEXPR_1-2_Hef1A-eYFP-4xFF1 and Tag-BFP, express yellow and blue fluorescent proteins. However, when co-transfected with U6 TetO: FF1 plasmid, FF1 miRNAs block the expression of yellow fluorescent proteins via binding to FF1 sites on pEXPR_1-2_Hef1A-eYFP-4xFF1; HEK293 cells therefore only appear blue (due to Tag-BFP). 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.

This experiment allowed us to characterize the ability of U6 promoter to drive the expression of short strands of RNA (like FF1 miRNA): by varying molar ratios of miRNA to pEXPR_1-2_Hef1A-eYFP-4xFF1 plasmid, we can calibrate the strength of the U6 promoter. Furthermore, we can harness such ability of U6 promoter to drive the expression of RNA oligonucleotides involved in processing or actuation. Another advantage to this system is that it can serve as an actuation output: the miRNA expressed can inhibit the expression of a gene of interest or relieve the inhibition of its expression.

Controlling mRNA Levels Using Hammerhead Ribozymes

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