Short RNA Production

To fully realize strand displacement as an in vivo solution, we need to be able to produce short strands of RNA inside the cells. We found the U6-TetO promoter, which does exactly that, and set out to test it. We ran an experiment using both constitutively expressed eYFP-4xFF1 and the U6TetO promoter driving FF1 production.

In this design, 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 express blue from 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.

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.