Our NOT Gate Design, Simulations and Optimization

Design

The NOT gate (signal inverter) design we eventually used comprises of multiple double- and single-stranded species. The most important of these are NOT gate (A), output (B) and buffer (C) complexes.
There are two important cases to consider: first the case where there is no input present (in digital terms, the input is 0), and second the case where there is a saturating level of input (digitally represented as a 1). We aim to convert the 0 into a 1 and the 1 into a 0 - the basic operation of a NOT gate. Refer to the diagram below throughout the explanation.

Case 1: No input present -> high signal
Here, the output strand (B) reversibly reacts with the NOT gate (A). The reaction is reversible because the output strand only displaces the green section on the NOT gate, but not further, meaning that the reaction could go back as well. This reversible reaction allows the output strand to react to a downstream buffer and amplifier to produce a high signal (a new nucleic acid strand is released and now active).

Case 2: Input present -> low signal
Here, the input strand and output strand both react with the NOT gate. The input strand displaces up to the pink region and the output strand displaces up to the green region. This is irreversible, as once strand displacement happens from both sides, the complex separates into two double-stranded species and there is no toehold to initiate the reverse reaction. Now the output strand is stuck and cannot be buffered and amplified to produce a signal (no new nucleic acid strand is released).

Diagram of our NOT gate. See the paragraph above for a more detailed description.

Optimization

In Vitro Results for Our NOT GATE

To test the functionality of the NOT gate as expressed by its transfer function (relating the input levels to output signal strengths) we performed in vitro studies with DNA as the nucleic acid for strand displacement. The output signal strand was fed to a reporter complex. This reporter, when triggered by a signal, will fluoresce in the red channel. Our experiments involved measuring these fluorescence levels for various amounts of initial input levels.

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 the 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 affects the kinetics of reactions involving an input signal and the gate:output as this reaction now changes from a tri-molecular reaction to a bi-molecular reaction. To solve this problem, we incorporate Hammerheads in the gate:output transcript. These same arguments and solutions apply for the threshold complex 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 and catalyzes its own cleavage.

Software rendering of the minimum free energy structure of the 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.

Our 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 constructs using the red fluorescent protein mKate.

The designs included 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.