Team:MIT/ResultsProcessing

From 2012.igem.org

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<h1>Overview</h1>
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<h1>Processing Overview</h1>
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<p>
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We demonstrated the first NOT gate compatible with the strand displacement system that we adopted.  The addition of a NOT gate will allow for more varied logic to be implemented using all forms of strand displacement, whether in vitro or in vivo, with RNA or DNA.
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Processing information involves using various logic gates to compute the value of a logic expression. Every such expression can be evaluated using a combination of AND, OR and NOT logic gates, making these three gates crucial. Qian <em>et al.</em> already designed and demonstrated AND and OR gates, however did not build a modular NOT gate.
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We also introduced the hammerhead ribozyme, a powerful RNA-cutting tool, to iGEM and the parts registry.  We intend to use the hammerhead ribozyme to manufacture RNA strand displacement gates in vivo.
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We demonstrated the first NOT gate compatible with toehold-mediated strand displacement in an <em>in vitro</em> setting using DNA. This NOT gate is modular <em>in vivo</em> thanks to production and degradation of RNA circuits.
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<div class= "section" id="m2bio">
 
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<h1>Not Gate In Vitro</h1>
 
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<p><img src="http://2012.igem.org/wiki/images/9/9d/NOT_GATE2.png" width=600/>
 
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<br> <i>Figure 1 - DNA molecules that constitute the NOT GATE </i>
 
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The original strand displacement paper demonstrated AND and OR gates, but did not include NOT gates.  We designed, built, and successfully tested a strand displacement NOT gate in vitro, expanding the computational structures possible with strand displacement.
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We also introduced the Hammerhead ribozyme, a powerful self-cleaving RNA structural motif, to iGEM and the parts registry. This ribozyme can be used to produce the double-stranded RNA complexes required for processing <em>in vivo</em>. <!-- TODO: "Read more about how Hammerheads can be used for strand-displacement controlled actuation."? -->
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<div class="section" id="NOTdesignbio">
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<h1>Our NOT Gate Design, Simulations and Optimization</h1>
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<h2>Design</h2>
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The design of our NOT gate is in Figure 1 above, where a letter with a '*' depicts a complementary domain to the one denoted by the letter alone. We arrived to this design after iterating through numerous other ideas, trying each time to reduce the number of molecules involved and their complexity.
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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.<br/>
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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.
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To understand the behavior of this NOT gate, it can be useful to consider two extreme cases: no input and saturation-level input.
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<strong>Case 1: No input present -&gt; high signal</strong><br/>
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Here, the output strand (B) <em>reversibly</em> 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).
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When the input is not present, molecule B can bind reversibly with A (by partially displacing a1) and reversibly with C. When B displaces c2 from C, molecule D frees the B strand that, consequently, is able to displace c2 from other C molecules.  c2 then triggers the readout by irreversibly displacing e2 from e1 (Therefore the role of D is to make B catalytic allowing it to react with more C molecules, amplifying the output). Consequently we will see high fluorescence.
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<strong>Case 2: Input present -&gt; low signal</strong><br/>
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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 <em>irreversible</em>, 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).</p>
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When the input is present in high concentration, B binds to a2, partially displacing a2 from a1. The input then binds to a1, completing what B started by fully and irreversibly separating a2 and a1. This step was inspired by the mechanism of the cooperative hybridization (Cooperative Hybridization of Oligonucleotides,David Yu Zhang,JACS 2011). Since B is stuck with a2, it can no longer displace c2 from C, and the readout pathway described above cannot continue. Consequently e2 cannot be displaced from the readout. Therefore we will see no fluorescence.
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<img src="http://2012.igem.org/wiki/images/9/9d/NOT_GATE2.png" alt="Our NOT Gate Design"><br/>
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<em>Diagram of our NOT gate</em>. See the paragraph above for a more detailed description.
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<h2>Optimization</h2>
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The figure below shows experimental validation of our NOT gate design.  As predicted, the concentration of the output strand decreases as the concentration of the input strand increases.
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<!-- TODO: all of the story needs to be written!!! -->
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<div class="section" id="NOTinvitrobio">
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<h1><em>In Vitro</em> Results for Our NOT GATE</h1>
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<img src="http://2012.igem.org/wiki/images/0/06/NOT_gate_small2.png"/>
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To test the functionality of the NOT gate as expressed by its transfer function (relating the input levels to output signal strengths) we performed <em>in vitro</em> 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.
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<br> <i> Figure 2 - Experimental verification of the in vitro NOT gate.  Output levels were measured through the displacement of a ROX-RQ strand, the e1 and e2 "readout" molecule in the header diagram.  As c2 displaces e2 from e1, the RQ quencher on e2 separates from the ROX fluorescent molecule on e1, and the ROX fluoresces.</i>
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One important consideration in implementing the NOT gate is the relative concentration of A with respect to input and B. If the concentration of A is too low, the cooperative hybridization between A, B, and a high concentration of input  can be slow.  In that case, B is free to displace c2 from C, triggering the output although the input level is high. On the other hand, if the concentration of A is too high, even without the presence of input, B will continuously reversibly bind with A.  Consequently, B is not available to displace c2 from C (in a reasonable time), and therefore we would not see a high level of output even when the level of input is low.
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In addition to the relative concentration of the different components, another important point is the absolute concentration of them. This is mainly due to the equilibrium thermodynamics of cooperative hybridization. Since there are three reactants, but only two products, at low concentration the reactants are more favorable in the reaction, whereas at high concentration the products will be more favorable.  
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In light of these considerations, we tuned the NOT gate first by finding a set of concentrations that give the correct qualitative behavior, and then by fine-adjusting A for the right trade-off between the interaction of A, input, and B when the input is high, and the interaction of A and B when the input is low.
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<!-- TODO: graphs!!! -->
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<h1>Not Gate Modeling</h1>
 
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<p> Creating a simulation helped us to find the right trade off, as mentioned before, in the choice of relative concentration of A with to respect of B and input. With this model, we were able to compute a transfer function for the NOT gate, which predicts the output levels produced in response to various input levels.</p>
 
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<p> The performance of the NOT Gate was analyzed using <a href = "http://lepton.research.microsoft.com/webdna/">Visual DSD</a>, an external software developed to model the kinetics of DNA strand displacement. Download the code for this simulation <a href = "http://2012.igem.org/File:NOTgate_MIT2012.txt">here</a>.
 
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<p><img src="http://2012.igem.org/wiki/images/6/65/NOT_gate_in_vitro_vs_simulation_small.png"/>
 
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<br> <i>Figure 3 - NOT GATE transfer function in vitro and by simulation using the software Visual DSD</i>
 
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Figure 3 shows the overlay of the simulated transfer function and the <i>in vitro</i> transfer function, subtracting the basal fluorescence. The graph demonstrates that the <i>in vitro</i> modeling accurately predicted the behaviour of the NOT gate. Note the negative slope, characteristic of NOT logic. Rate constants for this simulation were based on the findings of the article "Scaling Up Digital Circuit Computation with DNA Strand Displacement Cascades, Lulu Qian  and Erik Winfree, <a href = "http://www.sciencemag.org/content/332/6034/1196/suppl/DC1">Science, 2011</a>".
 
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<h1>Not Gate Optimization</h1>
 
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<p><img src="http://2012.igem.org/wiki/images/5/5c/MIT2012_NOT_gate_optimization_medium.png"/>
 
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<br> <i>Figure 4 - NOT gate transfer function for different concentration of constitutive molecules</i>
 
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As mentioned before, we arrived at the NOT gate transfer function depicted in Figure 2 after many attempts to find a working set of strand concentrations, and after that, to fine-tune the behavior.
 
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<p> In Figure 4, the effect of relative and absolute concentrations on the some transfer function of the NOT gate can be seen. For each transfer function, B is at x/2, C and readout are at 1x, and D is at 2x.  With the term 'absolute concentration' we mean changing the value of x, that is, the concentration of each molecule.  With the term 'relative concentration' we mean the change of only the concentration of the A molecule.
 
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When, for instance, x = 8nM and A is at 1x, the transfer function is very far from NOT gate behavior.  Moreover, when  we increased A to 2.5x (keeping everything else the same) to decrease the level of output for high levels of input, we actually had a level of output that was even higher.
 
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When instead we increased x at 16.5nM, we start to see a transfer function with a behavior much closer to that of a NOT gate.  The best absolute concentration appears to be x = 20nM.  Using this value of x, setting A to 1.4x gives the best discrimination between high and low output.
 
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<p><img src="http://2012.igem.org/wiki/images/c/c5/MIT2012_NOT_gate_10x_simulation_small_%281%29.png"/>
 
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<br> <i>Figure 5 - NOT gate transfer function simulation with A at 10x.</i>
 
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<p>The transfer function with sigmoidal behavior in Fig5 still refers to our NOT GATE (that is the one depicted in fig1). <br> The only difference is the fact that the molecule A in this case is at 10x.
 
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<br> We ran an in vitro experiment with A at 10x, but this particular setting let us have a low level of output when the input level was low, even after a long time.
 
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One possible explanation can be the fact that, in the annealing of A, the strand a2 was in excess. Therefore increasing the concentration of the molecule A with respect to B lets B improperly reacts with a2. Consequently, B could no more displace c2 from C. We have already planned to run new experiments with new annealing for the molecule A.
 
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<div class= "section" id="m3bio">
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<h1>Hammerhead Ribozymes</h1>
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<h1>Hammerhead Ribozymes for Producing RNA Circuits <em>In Vivo</em></h1>
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<h2>Motivation</h2>
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The hammerhead ribozyme is a self-splicing ribozyme: upon being transcribed to RNA, it folds into a particular secondary structure and catalyzes its own cleavage.
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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.
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<h2>Background: Hammerhead Ribozymes</h2>
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Hammerheads may allow us to transcribe gate complexes in vivo in a single transcript. The DNA coding for the gate-output complex would include the sequences for each strand, separated by hammerheads. When this DNA is transcribed into RNA, the gate and output sequences bind by Watson-Crick base-pairing, and the hammerhead folds and cleaves, resulting in the correct double-stranded gate.
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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.
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<a href="http://2012.igem.org/wiki/images/7/78/N117-mfe.png"><img src="http://2012.igem.org/wiki/images/b/bb/N117-mfe-small.png" alt="Software rendering of the minimum free energy structure of the N117 Hammerhead ribozyme"/></a>
<a href="http://2012.igem.org/wiki/images/7/78/N117-mfe.png"><img src="http://2012.igem.org/wiki/images/b/bb/N117-mfe-small.png" alt="Software rendering of the minimum free energy structure of the N117 Hammerhead ribozyme"/></a>
<a href="http://2012.igem.org/wiki/images/c/ce/N117.png"><img src="http://2012.igem.org/wiki/images/5/50/N117-small.png" alt="Software rendering of the minimum free energy structure of the folded hammerhead ribozyme"/></a><br/>
<a href="http://2012.igem.org/wiki/images/c/ce/N117.png"><img src="http://2012.igem.org/wiki/images/5/50/N117-small.png" alt="Software rendering of the minimum free energy structure of the folded hammerhead ribozyme"/></a><br/>
<em><a href="http://nupack.org">Software</a> rendering of the minimum free energy structure of the Hammerhead ribozyme</em>. Left: abstract ball-and-chain representation, right: 3D rendering of RNA. The left side of each diagram represents the 5' end of the ribozymes.
<em><a href="http://nupack.org">Software</a> rendering of the minimum free energy structure of the Hammerhead ribozyme</em>. Left: abstract ball-and-chain representation, right: 3D rendering of RNA. The left side of each diagram represents the 5' end of the ribozymes.
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<h2>Our Design</h2>
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<h2>Hammerheads for Producing Gate:Output Complexes</h2>
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Hammerheads may allow us to transcribe gate:output double-stranded complexes <em>in vivo</em> 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.
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<img src="http://2012.igem.org/wiki/images/e/e1/Hammerhead.png" />
<img src="http://2012.igem.org/wiki/images/e/e1/Hammerhead.png" />
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<i>Using hammerheads to make gate-output complexes in vivo. The initial transcript contains the gate and output strands, separated by hammerheads and a spacer sequence. The RNA folds through base pairing, allowing the gate and the output to bind together, and the hammerhead structure to form.  Once folding is complete, the hammerheads self-cleave, revealing the final gate-output complex.</i>
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<em>Using Hammerheads to make gate:output complexes </em>in vivo. See the above paragraph for a detailed description.
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<h2>Hammerheads as RNA-compatible Actuators</h2>
<h2>Hammerheads as RNA-compatible Actuators</h2>
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We are currently working on producing a mutant hammerhead to validate our preliminary results of hammerhead-mediated mRNA regulation.
We are currently working on producing a mutant hammerhead to validate our preliminary results of hammerhead-mediated mRNA regulation.
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Revision as of 17:50, 26 October 2012

DEPRECATED. DO NOT USE OR EDIT. If a page uses this template, relink with MIT-results2.

Overview

Processing information involves using various logic gates to compute the value of a logic expression. Every such expression can be evaluated using a combination of AND, OR and NOT logic gates, making these three gates crucial. Qian et al. already designed and demonstrated AND and OR gates, however did not build a modular NOT gate.

We demonstrated the first NOT gate compatible with toehold-mediated strand displacement in an in vitro setting using DNA. This NOT gate is modular in vivo thanks to production and degradation of RNA circuits.

We also introduced the Hammerhead ribozyme, a powerful self-cleaving RNA structural motif, to iGEM and the parts registry. This ribozyme can be used to produce the double-stranded RNA complexes required for processing in vivo.

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).

Our NOT Gate Design
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 N117 Hammerhead ribozyme Software rendering of the minimum free energy structure of the folded hammerhead ribozyme
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.