Decoys and Tough Decoys (TuDs)

Decoy and Tough Decoy overview

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.

Double Repression System Design

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.

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.

Source: Kitamura 1998.

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.

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 was more relieved, resulting in higher intensity.

Ultimately in increasing the relative concentration of decoy that was transfected, we observed a slight relief of repression of the system. We are currently working on better optimizing our transfection ratios so that our system can be tightly regulated and have a defined switching behavior between on and off states for protein expression. We also plan to use decoys or tough decoys as conditional outputs of upstream strand displacement that could be used to trigger protein expression in specific cases.

Conclusion

Through our testing of decoys and TuDs, we demonstrated induction of downstream protein effects through the RNA strand displacement motif. We can change levels of protein expression by interacting with the RNA pathways by which they are produced; either through repression or double repression systems at the protein translation level.

Hammerhead Ribozymes for Producing RNA Circuits In Vivo

Motivation

Producing RNA strand-displacement circuits inside cells is non-trivial, as the gate:output complexes in these circuits need to be properly base-paired as soon as they are produced. Otherwise, a loose output strand might trigger a downstream gate:output or simply anneal with an incorrect gate. This problem can be removed by producing gate:output complexes as single transcripts that then fold to form a stem-loop type structure. This poses a second problem, since this in turn affects the kinetics of reactions involving an input signal and the gate:output, as this reaction now changes from a tri-molecular reaction (involving the input, gate and output strands) to a bi-molecular reaction (now the gate:output complex is just one strand). To solve this problem, we incorporate Hammerheads in the gate:output transcript to cut the gate:output transcript into two.
These same arguments and solutions apply for the threshold complex (introduced by Qian et al.) as well.

Background: Hammerhead Ribozymes

The Hammerhead ribozyme is a self-cutting ribozyme: upon being transcribed to RNA, it folds into a particular secondary structure which enables it to catalyze its own cleavage.

Software rendering of the minimum free energy structure of the N117 Hammerhead ribozyme. Left: abstract ball-and-chain representation, right: 3D rendering of RNA. The left side of each diagram represents the 5' end of the ribozymes. The N117 Hammerhead sequence was derived from Yen et al. (2004).

Design

Hammerheads may allow us to transcribe gate:output double-stranded complexes in vivo in a single transcript. The DNA coding for the gate:output complex would include the sequences for each strand, separated by sequences coding for the Hammerhead RNA. When this DNA is transcribed into RNA, the gate and output sequences bind by Watson-Crick base-pairing, while the Hammerhead folds and cleaves within its sequence, resulting in the correct double-stranded gate:output two-strand complex. See below for the schematic of this process.

Using Hammerheads to make gate:output complexes in vivo. See the above paragraph for a detailed description.

Hammerheads as RNA-compatible Actuators

Additionally, Hammerheads may serve as an actuation mechanism compatible with RNA machinery. In particular, Hammerhead cleavage can destabilize mRNAs, resulting in decreased mRNA levels, which reduces protein levels (Yen et al., 2004). We used the Hammerhead sequences by Yen et al. (in particular, N117) and designed a family of Hammerhead-containing constructs using the red fluorescent protein mKate.

The designs include putting the Hammerhead motif in the 5' or 3' UTR of mKate, producing either Hammerhead-mKate (HH-mKate) or mKate-Hammerhead (mKate-HH) constructs. For HH-mKate, the Hammerhead was placed before the Kozak sequence and a spacer was included to ensure that the Hammerhead forms correctly (validated using NUPACK). For mKate-HH, the Hammerhead sequence was placed after the stop codon. Again, the folding of the Hammerhead structure was confirmed in simulations.

Circuits to test Hammerhead ribozyme function in vivo. The Hammerhead in the circuit on the right should self-cleave post transcription, destabilizing the mKate mRNA and preventing its expression. Here, the HH-mKate construct is depicted.

Key: Red: mKate, Blue: mKate-Hammerhead, Green: Hammerhead-mKate.
100,000 HEK293 cells were transfected with equimolar amounts of Hef1a:TagBFP, as a transfection marker, and one of the following: Hef1a:mKate, Hef1a:Hammmerhead-mKate, and Hef1a:mKate-Hammerhead. 48 hrs later, cells were harvested and analyzed by flow cytometry until 10,000 events were recorded. The histograms are gated on the blue population of cells, meaning that we are examining red fluorescence (mKate signal) in cells that also received the TagBFP DNA. There is less red fluorescence in the Hammerhead constructs compared to the Hef1a:mKate. This suggests that the Hammerhead ribozymes could be cleaving the mRNA.

We are currently working on producing a mutant Hammerhead to validate our preliminary results of Hammerhead-mediated mRNA regulation.