Decoys and Tough Decoys (TuDs)

We designed a proof-of-concept circuit to test the in vivo functionality of decoy and tough decoy (TuD) RNA, a mechanism of conditional output actuation that could interface with our upstream RNA strand displacement cascade.

To develop a mechanism of actuation for our circuitry, we considered using a tight RNA interference-based double repression system that could be characterized by a distinct switch between the on and off states. We investigated the use of decoy and tough decoy (TuD) RNA, which were novel RNA interference technologies inspired by Haraguchi et. al. (Nucleic Acids Res. 2009). This mechanism would allow for conditional expression of our desired output, a yellow fluorescent protein, based on the informational processing assessment characterized by the upstream RNA strand displacement cascades.

Decoy RNA and TuD RNA serve as novel RNA interference technologies that can relieve miRNA repression of a particular gene. In accordance with the central dogma of biology, transcribed mRNA strands are translated by cellular machinery into proteins. Mammalian cells incorporate RNA interference technologies to regulate gene expression through the presence of miRNA strands, which can complementarily bind to regions of mRNA and suppress the translation into a protein. Decoy and TuD RNA work to relieve this repression, by complementarily binding to regions of miRNA to inhibit their repressive functions. While decoy RNAs are merely single strands of RNA with miRNA-binding domains, TuD RNAs incorporate a stabilizing stem-loop structure, with two antisense miRNA-binding domains that can allow for additional miRNA coordination. We observed from the Haraguchi et. al. study that the particular TuD RNA structure yielded increased levels of activity compared to decoy RNA, so we attempted to assess the functionalities of both designs.

Sources: Haraguchi et al. 2009, Kitamura 1998.

Furthermore, the studies showed that a small 4-nucleotide bulge sequence in the miRNA-binding domains of decoy and TuD RNA increased their abilities to relieve repression of protein expression. The bulge in the miRNA-binding sequence likely assisted the decoy and TuD RNA in disrupting RISC complex activity in mammalian cells. This characteristic informed our decision to test two different decoy and TuD RNA species – one that lacked a 4-nucleotide bulge in the miRNA-binding domain, and one that had the bulge sequence.

Circuit Design

In order to assess the in vivo functionality of decoy and TuD RNA, we designed a proof-of-concept double repression circuit that would be implemented and tested in mammalian cells. We tested different parameters to characterize the functionality of the different components. Our circuit begins with a constitutively (always) expressed yellow fluorescent protein, with the mRNA transcript containing several binding domains for a particular miRNA. We then introduce the miRNA into the system, resulting in repression of the protein expression. Finally, upon introducing decoy and TuD RNA into the system, the repression is subsequently relieved, allowing for expression of the yellow fluorescent protein.

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.

We tested two different ratios of eYFP, eYFP intronic mKate, and decoy. The first had a ratio of 1:1:1 and the second had 1:1:2. In the instance with more decoy, the FIT-C yellow population shifted to much higher intensity.

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:

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.

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.

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.