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: http://nar.oxfordjournals.org/content/early/2009/02/17/nar.gkp040.abstract
http://www.ncbi.nlm.nih.gov/pubmed/9695408

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.

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.