  <meta charset='utf-8'>
  <meta http-equiv="X-UA-Compatible" content="IE=edge">
  <meta name="viewport" content="width=device-width, initial-scale=1">
  <link rel="stylesheet" href="styles.css">
  <script src="http://code.jquery.com/jquery-latest.min.js" type="text/javascript"></script>
  <script src="script.js"></script>
  <title>Breaking RNA</title>

<style>

column-one,.toc {

 display: none;

} body.mediawiki {

 background: url(http://openwetware.org/images/8/8c/Breaking_RNA_Background.png);
 background-size: cover;

}

globalWrapper{

 width: 80%;
 margin-left: 10%;
 position: absolute;

}

content{

 margin: 0px;

 background: white;

}

h1,h2,h3,p,ul,li {

 font-family: 'Open Sans', sans-serif;
 font-weight: 300;
 color: Black;
 text-align: justify;

} h1

  {
   text-align: center;
  }

h3

  {
   font-size: 20px;
   position: relative;
   text-align:center;
  }

p

  {
   font-size: 16px;
   line-height: 30px;
   padding: 10px 150px;
  }

objectives

  {
  }

objectives ul

  {
   padding: 0 0 0 30%;
   margin: 0 auto;
  }

objectives li

  {
   font-size: 18px;
   list-style: none;
   line-height: 30px;
  }

submenu

  {
   margin: 0px 40px 0px 22px;
  }

submenu a

  {
   color: black;
   text-decoration: underline;
  }

submenu a:hover

  {
   color: rgba(255,255,255,0.99);
  }

submenu ul

  {
   text-align: center;
   padding: 8px 0px;
   border: 3px solid black;
   background: rgba(70,150,70,0.8);

<!--background: url(http://openwetware.org/images/8/8c/Breaking_RNA_Background.png);

   background-size: cover;-->

submenu li

  {
   font-size: 18px;
   color: black;
   padding: 0px 20px;
   display: inline-block;
  }

supplemenu

  {
   margin: 0px 40px 0px 22px;
  }

supplemenu a

  {
   color: black;
   text-decoration: underline;
  }

supplemenu a:hover

  {
   color: rgba(255,255,255,0.99);
  }

supplemenu ul

  {
   text-align: center;
   padding: 15px 0px;
   border: 3px solid black;
   background: rgba(70,150,70,0.8);

supplemenu li

  {
   font-size: 18px;
   color: black;
   padding: 0px 10px;
   display: inline-block;
  }

floatmenu

  {
   position: fixed;
   bottom: 0;
   width: 78%;
   font-size: 18px;
   color: black;
   padding: 0px 10px;
   background: rgba(70,150,70,1);
   border: 3px solid black;
  }

floatmenu td

  {
   text-align: center;
   height: 36px;
  }

floatmenu td:hover

  {
   background: white;
  }

button

  {
   border-left: 3px solid black;
  }

floatmenu a

  {
   text-decoration: none;
   color: black;
  }

@import url(http://fonts.googleapis.com/css?family=Open+Sans:700);

#cssmenu {

 background: url(http://openwetware.org/images/8/8c/Breaking_RNA_Background.png);
 background-size: cover;
 width: auto;
 border: 3px solid rgba(0,0,0,0.8);

} #cssmenu ul { list-style: none; margin: 0; padding: 0; line-height: 1;

         text-align:center;

display: block; zoom: 1; } #cssmenu ul:after { content: " "; display: block; font-size: 0; height: 0; clear: both; visibility: hidden; } #cssmenu ul li { display: inline-block; padding: 0; margin: 0;

         line-height: 1;

} #cssmenu.align-right ul li { float: right; } #cssmenu.align-center ul { text-align: center; } #cssmenu ul li a { color: #ffffff; text-decoration: none; display: block; padding: 15px 25px; font-family: 'Open Sans', sans-serif; font-weight: 300; text-transform: uppercase; font-size: 14px; position: relative; -webkit-transition: color .25s; -moz-transition: color .25s; -ms-transition: color .25s; -o-transition: color .25s; transition: color .25s; } #cssmenu ul li a:hover { color: #f2ff00; } #cssmenu ul li a:hover:before { width: 100%; } #cssmenu ul li a:after { content: ""; display: block; position: absolute; right: -3px; top: 19px; height: 6px; width: 6px; background: #ffffff; opacity: .5; } #cssmenu ul li a:before { content: ""; display: block; position: absolute; left: 0; bottom: 0; height: 3px; width: 0; background: #f2ff00; -webkit-transition: width .25s; -moz-transition: width .25s; -ms-transition: width .25s; -o-transition: width .25s; transition: width .25s; } #cssmenu ul li.last > a:after, #cssmenu ul li:last-child > a:after { display: none; } #cssmenu ul li.active a { color: #f2ff00; } #cssmenu ul li.active a:before { width: 100%; } #cssmenu.align-right li.last > a:after, #cssmenu.align-right li:last-child > a:after { display: block; } #cssmenu.align-right li:first-child a:after { display: none; } @media screen and (max-width: 768px) {

         #supplemenu ul li {

float: none; display: block; } #supplemenu ul li a { width: 100%; } #cssmenu ul li { float: none; display: block; } #cssmenu ul li a { width: 100%; -moz-box-sizing: border-box; -webkit-box-sizing: border-box; box-sizing: border-box; border-bottom: 1px solid #ffffff; } #cssmenu ul li.last > a, #cssmenu ul li:last-child > a { border: 0; } #cssmenu ul li a:after { display: none; } #cssmenu ul li a:before { display: none; } }

</style> </head> <body> </body> </html> EDIT

Motivation and approach

Many cellular processes behave like circuits. Since the discovery of the Lac Operon in 1961 [1], a myriad of circuit-like systems have been shown to exist in a wide variety of organisms. Many synthetic biologists have taken advantage of this concept to engineer cellular activity and achieve new functions [2, 3]. However, most mechanisms involve thousands of unknown regulatory pathways. This complexity hampers our ability to predict and program new biological functions. Our project focuses on designing two canonical biological circuits, a switch and a clock, in an in vitro environment. In contrast to in vivo systems, in vitro circuits can be better controlled and understood using mathematical models, and can be tuned more easily to produce desired effects. We use few biological components (DNA, RNA and few proteins) to design a switch and a clock that are regulated through aptamers, and we characterize the main reactions required for their operation.

In other existing transcriptional synthetic circuits, regulatory interactions between genes were built using proteins or RNA that recognize and alter the conformation of the promoter region of another gene, thus modulating its activity [4]. We take an alternative approach: we use RNA aptamers modulate the activity of enzymes, rather than targeting promoters. Thus, aptamers create the feedback loops necessary to build our circuits. RNA is unique in that it can be an information-carrying polymer (such as mRNA) or a functional molecule (such as ribozymes and aptamers). RNA functions are mediated by its ability to fold into tertiary structures, of which aptamers are an example[5]. For our project, we use RNA aptamers that were evolved to inhibit T7 RNA polymerase and SP6 RNA polymerase[6, 7]. However, currently there are no established methods to reverse aptamer binding and inhibition in a rational manner. Reversible inhibition and repression are necessary to build dynamic circuits. Therefore, our first objective is to design and characterize DNA and RNA aptamer complements, which can reactivate the enzymes by removing the bound aptamers. We call these re-activating strands "Kleptamers" (from the Greek verb klepto = 'to steal'; Kleptamer ='strand that steals away the aptamer'). To monitor the dynamics of our circuits we also design and characterize a multifunctional molecular beacon which be will be an active part of the final circuits and at the same time will act as a reporter.

To build our circuits we integrate domain-level strand design, modeling, and experiments in vitro. Only few in vitro toolkits currently exist to program molecular dynamic circuits like clocks and switches [7, 8], and our project aims at expanding these toolkits to include aptamers.

Objectives

To achieve our overall goal of building aptamer-based circuits we pursued these objectives:

1) Design of the circuits and mathematical modeling
2) Characterization of activation and inhibition reactions
3) Assembly of the circuits.

EDIT

1. Design of the circuits and mathematical modeling

General approach description.

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.

Switch

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.

Clock

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.

2. Activation and inhibition reactions

Inhibition reactions: Aptamer Design

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[6, 7]. 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.

Activation reactions: "Kleptamer" Design

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!

Secondary structure of RNA aptamer when bound to SP6 RNA Polymerase.

Thus, it is important to take advantage of the differences in the secondary structure of the unbound and bound forms of the RNA aptamer.

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.

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.

3. Assembly of Circuits

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.

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.

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:

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.

References

All Medline abstracts: PubMed | HubMed

Biomod/2014/UCR/Breaking RNA/Project

Contents

Motivation and approach

Objectives

To achieve our overall goal of building aptamer-based circuits we pursued these objectives:

1. Design of the circuits and mathematical modeling

Switch

Clock

2. Activation and inhibition reactions

Inhibition reactions: Aptamer Design

Activation reactions: "Kleptamer" Design

3. Assembly of Circuits

References

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

research

Tools