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| {{UCR/Breaking-RNA/HEAD}}
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Project&action=edit EDIT]
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| <p></p>
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| <h1>Motivation</h1>
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| <p>Biological circuitry plays a pivotal role in proper cell functioning. Since the elucidation of the Lac Operon in 1961<cite>p1</cite>, a myriad of circuit-like operons have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of the concept of an operon to manipulate cellular activity<cite>p2 p3</cite>. However, most mechanisms involve thousands of unknown regulatory pathways. This ambiguity severely impedes the ability to reliably predict and program cellular function. Our project focuses on designing biological circuits ''in vitro'' which in contrast to ''in vivo'' systems, can be clearly understood by mathematical modeling and be tuned to produce desired effects. Using basic biological components (RNA Polymerase & Oligonucleotide sequences) we are attempting to design and implement programmable and reliable bistable and oscillatory circuits.</p>
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| <h1>Approach</h1>
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| [[Image:Approach.png|center|800px]]
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| <p>In order to develop complex circuitry, there must first be a wide set of tools to choose from. Thus, it's imperative that more ways to control biological components are developed. One approach is to use specific strands of oligonucleotides that can down regulate or up regulate transcription. In past, for establishing connection in a circuit, RNA transcribed form one gene has been used as to specifically recognize and interfere with promoter region of another gene<cite>p4</cite>. Here we aim to take a novel alternative approach. RNA is unique in that it has the ability to be used as an informative polymer, such as mRNA, or as a catalyst, such as ribozymes. This catalytic behavior is a result of its ability to readily fold into functional tertiary structures. One such example of functional RNA strands are aptamers<cite>p5</cite>. For this work, we use RNA aptamers that are known to inhibit T7 RNA polymerase and SP6 RNA polymerase<cite>p6 p7</cite>. But currently there are no established way to reverse this inhibition in a programmable way. So we aim to design and characterize DNA and RNA complements of aptamer, which can reactivate the enzymes by removing the aptamers bound to the enzymes. We call these re-activation strands "Kleptamers" (Klepto = 'stealing'; Kleptamer ='strand that steals away the aptamer'). In order to monitor the dynamics of the circuitry we also aim to design and characterize multifunctional molecular beacons which be would part of the functional circuit and at the same time will act as a reporter. We aim to put all together for the creation of a novel biological circuits such as bistable or oscillatory systems. We aim to test the topology of the bistable switch and the oscillator rigorously using mathematical modeling and use insights we obtained from modeling towards experimental implementation of these circuits. Our project helps to expand the toolbox of in-vitro synthetic biology. </p>
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| <h1>Objectives</h1>
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| <div id='objectives'>
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| <h3>Engineering biological circuits necessitates the integration of three crucial components:</h3>
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| <ul>
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| <li> 1) Design of the circuits and modeling </li>
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| <li> 2) Activation and inhibition reactions </li>
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| <li> 3) Assembly of the circuits. </li>
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| </ul>
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| </div>
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| <p></p>
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| [http://openwetware.org/index.php?title=Biomod/2014/UCR/Breaking_RNA/Project&action=edit EDIT]
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| <h1> <div id = "Design of the circuits and modeling"> 1. Design of the circuits and modeling </div></h1>
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| General approach description.
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| <p>Creating and designing simulations using computing programs, such as MATLAB, are important for testing the various mechanisms involved in the systems. This is particularly useful for coming up with preliminary conditions for in vitro experiments, as well as determining the robustness of the schematic.</p>
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| <h2> Switch </h2>
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| [[Image: SimpleSwitch.jpg|center|500px]]
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| Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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| <h2> Clock </h2>
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| [[Image: SimpleClock.jpg|center|500px]]
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| Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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| <h1><div id = "Activation and inhibition reactions"> 2. Activation and inhibition reactions </div></h1>
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| <h2><u>Inhibition reactions: Aptamer Design</u></h2>
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| <p>Specific inhibition of enzymatic activity is of major importance for our system. RNA aptamers that inhibit T7 RNA polymerase and SP6 RNA polymerase have been reported previously<cite>p6 p7</cite>. Among various sequence variants published in the original papers, we chose those reported to have higher binding affinity to RNA polymerases. We then designed genes to synthesize RNA aptamer transcripts . Then we proceeded to characterize the effectiveness of aptamer induced RNA polymerase inhibition in each case. </p>
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| <h2><u>Activation reactions: "Kleptamer" Design</u></h2>
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| <p>[[Image: KleptamerDesign.jpg|center|500px]]</p>
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| <p>Similar to inhibition, it is also crucial to allow the reactivation of the enzymes for oscillations. In order to reactivate the RNA polymerases, the RNA aptamers must be removed. We can do this by using a DNA strand that is complementary to the aptamer. The DNA complement will have a higher affinity to the RNA aptamer than does the enzyme, allowing the inhibitor to be "peeled" off from the enzyme and restoring transcription. It's important to ensure that the complement strand does not bind to the RNA aptamer not bound to the enzyme. If it does, there will be no chance for the aptamer to inhibit the RNA polymerase! </p>
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| [[Image: Oruchi.png|right|250px|thumb| <font size="1.5">Secondary structure of RNA aptamer when bound to SP6 RNA Polymerase.</font>]] [[Image: aptamer_solution.png|right|240px|thumb|<font size="1.5">Secondary structure of RNA aptamer when free in solution.</font> ]]
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| <p>Thus, it is important to take advantage of the differences in the secondary structure of the unbound and bound forms of the RNA aptamer. </p>
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| <p>The binding properties of the RNA aptamers rely on their secondary structure. For our strands, secondary structure is different when bound to the RNA polymerase than when unbound. Using NUPACK and Mori, ''et. al.'' we can make an estimation of how the DNA complement will interact with either of the two secondary structures.
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| Mori, ''et. al.'' were able to determine the secondary structure when attached to SP6 RNA polymerase and NUPACK was used to determine the structure of the unbound aptamer. Strands that are complement to the areas boxed in red are likely to have little interactions occurring, whereas, complements to nucleotides boxed in green will likely have high interactions. It is ideal that a strand is complement to most or all of the nucleotides that are boxed in order to get the most favorable interactions. A similar approach was taken for the T7 kleptamer design. </p>
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| <h1><div id="Assembly of Circuits">3. Assembly of Circuits</div></h1>
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| Now that the tools are designed, the next step is to begin piecing the system together to ensure each component is working properly. There are two main methods used to show evidence for our system, gel electrophoresis and fluorometry. Gel electrophoresis provides an excellent means of qualitatively determined the desired interactions are taking place. Our experiments pertain mainly to the use of 10% Polyacrylamide native gels. Since our system involves a dynamical aspect, this method is not sufficient. Fluorometry can be used in this case to determine the kinetics and the dynamics.
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| Unfortunately, we can't measure directly the activity of the enzymes. However, we can determine activity indirectly with the use of fluorescent dyes. [[Tsien, et. al.]] report the observation that specific aptamers can be used to switch on fluorescence of Triphenylmethane dyes, more specifically, Malachite green. Likewise, [[Jaffrey, et. al.]] have used SELEX to find an aptamer sequence that will bind to, and greatly enhance the fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolidine (DFHBI). These molecules are incorporated into the design as the reporter system. This is done by using genes that code for an aptamer that will bind to the fluorophore. In that way the RNAPs will code the aptamer which will bind to the fluorophore and increase fluorescence intensity! We have designated that DFHBI aptamer gene has an SP6 RNAP promoter, whereas Malachite green aptamer gene has a T7 RNAP promoter. Thus, it can be determined which enzyme is activated or inhibited by viewing the rate of fluorescence intensity increase for each respective fluorophore.
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| [[Image: Fluorescent_dye.png|400px||center|thumb|<font size="1.5"> A cartoon representation of the reporter system. The aptamer will interact with the dye, resulting in enhanced fluorescence of the molecule. </font>]]
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| Before the circuits can be assembled together to make a circuit, the individual components should be tested. This has two advantages. First, the circuit cannot function if even one portion does not work. By performing experiments on the components it ensures the system is plausible. Second, by performing individual reactions one at a time, the parameters used in the model can be better estimated to give more accurate approximations. There are five major portions that need to be tested before running the circuit. These include the following:
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| [[Image:Sp6 apt&sp6RNAP.png|left|300x175px|thumb|Inhibition of SP6 RNAP with R1]]
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| [[Image:Peel off aptamer.png|lright|300x175px|thumb|Reactivation of SP6 RNAP with K1]]
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| [[Image:R4aptamer.png|center|300x175px|thumb|Reactivation of SP6 RNAP with R4]]
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| [[Image:T7inhibitor.png|left|300x175px|thumb|Inhibition of T7 RNAP with R3]]
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| [[Image:R3 T7 R2.png|right|300x175px|thumb|Reactivation of T7 RNAP with R2]]
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| <br><br><br><br><br><br><br>
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| <p><font size="3.5">The aptamer encoded by T7 RNA Polymerase must be able to bind and inhibit to SP6 RNA Polymerase. This can be ascertained throughout the use of glee electrophoresis and fluorometry. Once this is determined, it's important to ensure that K1 can properly remove the aptamer from SP6 RNA Polymerase. This also was done through the use of gel electrophoresis and fluorometry. Another kleptamer involved in SP6 reactivation is strand R4 and was determined by gel electrophoresis only. are two mechanism that need to be verified for T7 RNAP. First, T7 needs to be able to self-inhibit itself via the transcription of G3. Finally, T7 must also be shown to be reactivated by the kleptamer R2. Both of these were verified using fluorometry and gel electrophoresis.Once all of the parts have been verified, the full oscillator can then begin to be implemented. Even at this point, however, it still can be very challenging since proper functioning can often times be very sensitive to initial conditions. Thus, models created using MATLAB will help to guide these experiments in order to help fine tune the initial concentrations of genes and enzymes to induce the proper parameters. Finally, the system is ready to begin full assembly. The model plays a crucial role at this point for giving approximations on the concentration of each component. However, the model will not be perfect and it will be necessary to perform tuning experiments. In this case, each concentration of each component will be varied one at a time. This will ensure that each concentration is optimized for the system to perform most efficiently. </font></p>
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| <h1>References</h1>
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| <biblio>
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| #p1 pmid=13718526
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| #p2 pmid=10659856
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| #p3 pmid=10659857
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| #p4 pmid=21921236
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| #p5 pmid=1697402
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| #p6 pmid=23650601
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| #p7 pmid=22426482
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| </biblio>
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