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. Through literature searches, we found the U6-TetO promoter, which does exactly that, and characterized it. We ran an experiment using both constitutively-expressed eYFP-4xFF1 and U6-TetO promoter-driven FF1 production.

In this design, HEK293 cells transfected with Hef1A:eYFP-4xFF1 and Hef1A:TagBFP express yellow and blue fluorescent proteins. However, when co-transfected with U6-TetO:shFF1 plasmid, FF1 shRNAs block the expression of yellow fluorescent proteins via binding to FF1 sites on eYFP-4xFF1 mRNA (mediated by RNA pathway proteins). The HEK293 cells therefore express only blue from Tag-BFP. As the amount of U6-TetO:shFF1 transfected increases from 0 to 160 fmol, 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.

Experimental results of our experiments for the U6 promoter and incorporating the RNAi pathway for an experimental readout. 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 amount of shFF1 increases from 0 to 160 fmoles, 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.

Change in the mean fold yellow fluorescence due to varying amounts of the U6-TetO:shFF1 construct. Here ,we plot the mean yellow fluorescence (FITC) of the various populations of the experiment described above.

These results show that we can control expression of short RNAs with the U6-TetO promoter. These RNA strands are essential to both our strand displacement information processing method and also to actuation of our computation into a tangible effect on the cell through protein modulation. The short RNA expressed can also inhibit the expression of a gene of interest or relieve the inhibition of its expression through Decoy or TuD interactions.

Hammerhead Ribozymes for Producing RNA Circuits In Vivo (in progress)

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 (Yen et al., 2004).

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.