Team:MIT/Actuation

From 2012.igem.org

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<div class= "section" id="TuDsDecoysbio">
<div class= "section" id="TuDsDecoysbio">
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<h1>Decoys and Tough Decoy (TuD) RNAs</h1>
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<h2>Background</h2>
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<p>
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Decoy and Tough Decoy (TuD) RNAs are novel technologies for regulating RNA interference (<a href="http://nar.oxfordjournals.org/content/early/2009/02/17/nar.gkp040.abstract">Haraguchi <em>et al.</em>, Nucleic Acids Res. 2009</a>). In short, they are single strands of RNA with one antisense microRNA binding domain (Decoy) or a stabilized stem-loop with two microRNA binding domains (TuD).<br/>
 +
In accordance with the central dogma of biology, transcribed mRNA strands are translated by cellular machinery into proteins. Mammalian cells incorporate a layer of expression control, RNA interference, to control mRNA levels. RNA interference uses microRNAs (miRNAs) that are processed by protein complexes to knock down mRNA levels in the cell, reducing protein expression. Decoys and TuDs are artificial strands of RNA with miRNA-binding domains that are thought to sequester the miRNA into stable complexes throuh complementary basepairing, disabling a particular RNA interference pathway.<br/>
 +
While miRNAs act as repressors, TuDs and Decoys act as double-repressors such that the presence of the TuDs/Decoys increases protein output.<br/>
 +
Thus miRNAs, Decoys and TuDs can potentially be incorporated in RNA strand-displacement circuits to control protein expression levels. As these are short RNAs, we imagine that these strands can be outputs of RNA strand displacement circuits. This will allow strand-displacement circuits to connect to traditional protein-based synthetic biology circuits.
 +
</p>
 +
<p>
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<img src="http://2012.igem.org/wiki/images/e/e2/Tud_circuit.png" width="600" alt="TuD / Decoy circuit"><br/>
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<em>Diagram depicting the interactions between Decoys, TuDs, miRNAs, mRNAs and proteins</em>. miRNAs are used in the RNA interference pathway to knock down mRNAs, which are usually translated into proteins. Decoys and TuDs repress this pathway, resulting in protein production.
 +
</p>
 +
 +
 +
<h2>Experimental Design</h2>
 +
<p>
 +
We designed a proof-of-concept double-repression circuit to test the <em>in vivo</em> actuation ability of Decoy and TuD RNAs as indicated by fluorescent protein expression.<br/>
 +
</p>
 +
<p>
 +
We observed from the Haraguchi <em>et al.</em> study that a particular TuD RNA structure, one with a stabilizing stem-loop structure, yielded increased levels of activity compared to Decoy RNA, so we set out to assess the functionalities of both Decoy and TuD designs. Furthermore, the studies showed that a small 4-nucleotide bulge sequence in the miRNA-binding domains of Decoy and TuD RNAs increased their abilities to relieve repression of protein expression. The bulge in the miRNA-binding sequence likely protected the Decoy and TuD RNA from the RISC protein complex. 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.
 +
</p>
 +
<p>
 +
We designed DNA plasmids containing the mammalian U6 promoter (see <a href="http://2012.igem.org/Team:MIT/CircuitProduction">Circuit Production</a> for more information on the U6 promoter) that could be used to drive expression of our RNAs in vivo upon transfection into mammalian cells.
 +
</p>
 +
<p>
 +
<img src="http://2012.igem.org/wiki/images/1/17/Tud_circuit22.png" width="600" alt="Circuit diagram for testing TuDs and Decoys"><br/>
 +
<em>Circuit diagram for testing TuD and Decoy-based actuation</em>. Our circuit begins with a constitutively (always; from the Hef1A promoter) expressed yellow fluorescent protein (eYFP), with the mRNA transcript containing several binding domains for a particular miRNA (FF4). We then introduce the miRNA (from the Hef1A-LacO:mKate-Intronic miR-FF4 construct) into the system, resulting in repression of yellow protein expression. Finally, upon introducing Decoy and TuD RNA into the system (the U6-TetO:Decoy FF4/TuD FF4 construct), the repression is subsequently relieved, allowing for expression of the yellow fluorescent protein. Note that the miR-FF4 miRNA is produced as a spliced-out intron from mKate, a red fluorescent protein. Thus red fluorescence indicates presence of miR-FF4.
 +
</p>
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<h1>Decoys and Tough Decoys (TuDs)</h1>
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<h2>Results</h2>
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<p><b>Decoy and Tough Decoy overview</b></p>
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<p>
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<p>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. </p>
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<img src="http://2012.igem.org/wiki/images/b/bb/Decoy.png" width="600" alt="Results from our experiments with Decoys"><br/><br/>
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<p><b>Double Repression System Design</b></p>
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<em>Flow cytometry data of our experiments with Decoy RNA.</em> We tested two different ratios of eYFP, mKate-Intronic miRFF4, and Decoy. The first experiment had a ratio of 1:1:1 and the second 1:1:2. In the instance with more Decoy the RNAi-induced repression was more relieved as indicated by a shift towards higher yellow fluorescence values in the FITC channel.
-
<p>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 <a href = "http://nar.oxfordjournals.org/content/early/2009/02/17/nar.gkp040.abstract">Haraguchi et al. (Nucleic Acids Res. 2009)</a>. 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. </p>
+
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<p>Decoy RNA and TuD RNA serve as novel RNA interference technologies that can relieve miRNA repression of a particular gene. In mammalian cells, RNA interference serve to regulate gene expression through 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. </p>
+
-
<img src="http://2012.igem.org/wiki/images/e/e2/Tud_circuit.png" width="600">
+
-
<p>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. </p>
+
-
<p><We first wanted to reproduce the results found by Haraguchi et al. in our lab, and so we designed four decoy and tough decoy RNA based upon successful parameters outlined by their studies. We designed DNA plasmids containing the mammalian U6 promoter that could be used to drive expression of our RNAs in vivo upon transfection into mammalian cells. </p>
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<p>Source: <a href = "http://www.ncbi.nlm.nih.gov/pubmed/9695408">Kitamura 1998</a>.</p>
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<p><b>Circuit Design</b></p>
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<p>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.  
+
</p>
</p>
-
<img src="http://2012.igem.org/wiki/images/1/17/Tud_circuit22.png" width="600">
+
<p>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 the on and off states for protein expression. We also plan to use Decoys or TuDs as conditional outputs of upstream strand displacement that could be used to trigger protein expression in specific cases.</p>
-
<p>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.
+
-
<img src="http://2012.igem.org/wiki/images/b/bb/Decoy.png" width="600">
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-
<p>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.</p>
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-
<h1>Conclusion</h1>
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<p>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.</p>
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</div>
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</div> <!-- /Decoys and TuDs -->
<div class= "section" id="Hammerheads">
<div class= "section" id="Hammerheads">

Revision as of 01:38, 27 October 2012

iGEM 2012

Overview

  • Results Overview

Circuit Production

  • Short RNA Production
  • Circuit Production: Hammerhead Ribozymes

NOT Gate

  • Design
  • Modeling
  • In Vitro Results

Sensing

  • Design
  • Modeling
  • In Vitro Results

Actuation

  • TuD and Decoy RNAs
  • Modulating Hammerheads

The Key Reaction

  • Design
  • Nucleic Acid Delivery
  • Experimental Strand Displacement

Our BioBricks

  • Favorites
  • All BioBricks

Attributions

  • Attributions

Decoys and Tough Decoy (TuD) RNAs

Background

Decoy and Tough Decoy (TuD) RNAs are novel technologies for regulating RNA interference (Haraguchi et al., Nucleic Acids Res. 2009). In short, they are single strands of RNA with one antisense microRNA binding domain (Decoy) or a stabilized stem-loop with two microRNA binding domains (TuD).
In accordance with the central dogma of biology, transcribed mRNA strands are translated by cellular machinery into proteins. Mammalian cells incorporate a layer of expression control, RNA interference, to control mRNA levels. RNA interference uses microRNAs (miRNAs) that are processed by protein complexes to knock down mRNA levels in the cell, reducing protein expression. Decoys and TuDs are artificial strands of RNA with miRNA-binding domains that are thought to sequester the miRNA into stable complexes throuh complementary basepairing, disabling a particular RNA interference pathway.
While miRNAs act as repressors, TuDs and Decoys act as double-repressors such that the presence of the TuDs/Decoys increases protein output.
Thus miRNAs, Decoys and TuDs can potentially be incorporated in RNA strand-displacement circuits to control protein expression levels. As these are short RNAs, we imagine that these strands can be outputs of RNA strand displacement circuits. This will allow strand-displacement circuits to connect to traditional protein-based synthetic biology circuits.

TuD / Decoy circuit
Diagram depicting the interactions between Decoys, TuDs, miRNAs, mRNAs and proteins. miRNAs are used in the RNA interference pathway to knock down mRNAs, which are usually translated into proteins. Decoys and TuDs repress this pathway, resulting in protein production.

Experimental Design

We designed a proof-of-concept double-repression circuit to test the in vivo actuation ability of Decoy and TuD RNAs as indicated by fluorescent protein expression.

We observed from the Haraguchi et al. study that a particular TuD RNA structure, one with a stabilizing stem-loop structure, yielded increased levels of activity compared to Decoy RNA, so we set out to assess the functionalities of both Decoy and TuD designs. Furthermore, the studies showed that a small 4-nucleotide bulge sequence in the miRNA-binding domains of Decoy and TuD RNAs increased their abilities to relieve repression of protein expression. The bulge in the miRNA-binding sequence likely protected the Decoy and TuD RNA from the RISC protein complex. 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.

We designed DNA plasmids containing the mammalian U6 promoter (see Circuit Production for more information on the U6 promoter) that could be used to drive expression of our RNAs in vivo upon transfection into mammalian cells.

Circuit diagram for testing TuDs and Decoys
Circuit diagram for testing TuD and Decoy-based actuation. Our circuit begins with a constitutively (always; from the Hef1A promoter) expressed yellow fluorescent protein (eYFP), with the mRNA transcript containing several binding domains for a particular miRNA (FF4). We then introduce the miRNA (from the Hef1A-LacO:mKate-Intronic miR-FF4 construct) into the system, resulting in repression of yellow protein expression. Finally, upon introducing Decoy and TuD RNA into the system (the U6-TetO:Decoy FF4/TuD FF4 construct), the repression is subsequently relieved, allowing for expression of the yellow fluorescent protein. Note that the miR-FF4 miRNA is produced as a spliced-out intron from mKate, a red fluorescent protein. Thus red fluorescence indicates presence of miR-FF4.

Results

Results from our experiments with Decoys

Flow cytometry data of our experiments with Decoy RNA. We tested two different ratios of eYFP, mKate-Intronic miRFF4, and Decoy. The first experiment had a ratio of 1:1:1 and the second 1:1:2. In the instance with more Decoy the RNAi-induced repression was more relieved as indicated by a shift towards higher yellow fluorescence values in the FITC channel.

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 the on and off states for protein expression. We also plan to use Decoys or TuDs as conditional outputs of upstream strand displacement that could be used to trigger protein expression in specific cases.

Using Hammerhead Ribozymes to Control Protein Expression

Motivation

Using the hammerhead ribozymes introduced in Circuit Production, we plan on controlling protein expression in vivo. We intend to use the self-cleaving action of hammerheads to control mRNA levels, and thus protein levels. Hammerheads are RNA constructs, so they should be able to interface with the RNA strands used in strand displacement. In particular, we imagine an RNA input strand binding to a hammerhead structure and destabilizing it, preventing the hammerhead from self-cleaving.

Design

We tested this idea by appending a hammerhead structure to the mKate fluorescent protein. The hammerhead sequence should cleave and destabilize the mKate transcript, preventing fluorescence. In addition, we tried adding short input strands complementary to the hammerheads. This should prevent the hammerhead from folding properly, restoring fluorescence. 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.