Team:Peking R/Project/RNAToolkit

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   <p class="notbookmaintitle" align=center>RNA toolkit<a name="start" id="start"></a></p>
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   <p class="notbookmaintitle" align=center>&nbsp;</p>
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   <p class="notbookmaintitle" align=center>&nbsp;</p>
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   <p class="notbookmaintitle" align=center>Overview</p>
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   <p>In recent years, RNA devices have emerged as powerful tools to regulate gene expression in vivo, and particularlyligand-responsive riboswitches/ribozymes enable us to manipulate translation  strength of specific genes upon different concentrations of ligandsLigand-responsive riboswitches/ribozymes regulate the translation rate of downstream gene by changing conformations, cleaving or splicing upon external  addition of ligand. Compared with transcriptional and post-translational  regulation, riboswitches/ribozymes function through allostery of RNA structurewhich requires little or no assistance from proteins, so the regulation  mechanism is relatively simpler and their functions are more decoupled from  native biological activities.</p>
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   <p>In order to fulfill the goal of establishing an extensible and versatile methodology for softcoding of genetic program, our team reached out to a great extent to search for available ligand-responsive riboswitches/ribozymes that meet our criteria and selected  them as our genetic rheostats.</p>
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<p>Candidates for genetic rheostats must meet  two basic criteria: firstly, they should possess a relatively plain dose-response curve, which would allow for precise translation strength modulation within a wide range of ligand concentration; secondly, ligands they recognize should be genetically and biochemically orthogonal to the host cellsin our case, <em>E.coli</em> cells, as much as  possible.</p>
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<p>Two candidates emerged as promising genetic rheostats that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive hammerhead ribozymes and theophylline-responsive riboswitches. By altering the upstream promoter and downstream coding sequence of the genetic  rheostats, we demonstrated that their performance was independent of sequence  context, which proved that our genetic rheostats are modular.</p>
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<p>To further extend the repertoire of our genetic rheostats, we created a ribozyme that functions with a different mechanism, which has an extreme low basal level. We substituted the aptamer domain of c-di-GMP group I intron to theophylline-responsive aptamer, thus invented a group I intron that senses theophylline to perform splicing function.</p>
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  <p align="center" class="TITLE2"><strong>High-performance RNA Controllers Regulated By TPP</strong></p>
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<p>Moreover, we introduced a general method to evolve genetic rheostat that senses a new ligand. By coupling an adenine  aptamer with hammerhead ribozyme and randomizing nucleotides in the linker domain, we evolved new genetic rheostat through dual selection, whose  self-cleavage could be regulated by adenine.</p>
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   <p class="mainbody"><a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1"></a>Just as  previously stated, even though proteins fulfill most of biological functions, such as enzymes as catalysts, receptors as signal transmitters, they have certain drawbacks when used as synthetic biology’s modules. One example is that they are often coupled with normal biological processes, so their functions depend much on the genetic context. RNA, as a large family of basic biomolecules, also possesses similar capacities to those of proteins.</p>
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   <p>Hammerhead ribozymes are small self-cleaving RNAs, first discovered in satellite RNAs of  plant viruses that catalyze a specific phosphodiester bond isomerization reaction in the course of rolling-circle replication <a href="#r101">[1]</a>. </a>More  recently a full-length hammerhead ribozyme from Schistosoma</em><em> mansoni</em> is being more frequently utilized for application. </a>As shown in <strong>Fig.1-A</strong>, this hammerhead ribozyme can  be truncated to a minimal, catalytically active motif consisting of three base-pairing stems (marked in colors) flanking a central core  of 15 mostly invariant nucleotides (marked in frame). And the conserved central  bases are essential for the hammerhead ribozyme’s catalytic activity<a href="#r102"> [1]</a>. <a name="OLE_LINK44" id="OLE_LINK44"></a><a name="OLE_LINK43" id="OLE_LINK43"></a><a name="OLE_LINK42" id="OLE_LINK42"></a><a name="OLE_LINK41" id="OLE_LINK41"></a>The tertiary structure shown in <strong>Fig.1-B</strong> indicates that the secondary structure of the<em> Schistosoma</em> hammerhead ribozyme can be distorted into a uridine turn because of distant loop/bulge interaction which induces changes in stem II while simultaneously  unwinding stem I. For the basic catalytic function of hammerhead ribozyme, the active site for self-cleaving of <em>Schistosoma</em> hammerhead ribozyme resides between stem III and stem I, as shown in <strong>Fig.1-A.</strong></p>
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  <p class="mainbody">It has been reported previously that mRNAs encoding enzymes involved in thiamine  (vitamin B1) biosynthesis in <em>Escherichia coli</em> can bind thiamine or its pyrophosphate</a> derivative without the assistance from protein cofactors <a href="#r203">[2]</a>. These ligand-binding mRNAs actually possess thiamine or pyrophosphate binding domaincalled aptamer, in which the binding event can bring about a conformational change  which is important for genetic control. This natural thiamine pyrophosphate  (TPP) aptamer can bind to TPP specifically and a defined structure is stabilized. As shown in <strong>Fig.1-C</strong>,  upon addition of TPP, TPP can bind loop in green through non-covalent bond</a>. <strong>Fig.1-D</strong> shows the tertiary structure of natural TPP aptamer binding to TPP.</p>
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    <td colspan="3" class="picturemark"><p align="left"><strong>Figure.1</strong></p>
 
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The  schematic structures of <em>Schistosoma </em>hammerhead</a> ribozyme and natural TPP aptamer. <strong>A) </strong>The  secondary structure of<em> Schistosoma</em> hammerhead ribozyme. Three base-pairing stems are shaded in colors. The part in  yellow represents stem III,  which is later modified to be the linker between hammerhead ribozyme and  aptamer. The part in blue represents stem II, and the purple and red ones stand  for two parts of stem I. The sequence in frame represents for the conserved  nucleotides. The red arrow points to the scissile bond. <strong>B)</strong> The tertiary structure of<em>Schistosoma</em> hammerhead ribozyme.  The cyan part indicates the fragment of mRNA after cleavage. The red one indicates  the active site for self-cleavage. The linker between hammerhead ribozyme and  aptamer is shown in yellow. <strong>C)</strong> The secondary structure of natural TPP aptamer. TPP  can bind to loop in green through non-covalent bond and the part marked in yellow indicates the linker  between hammerhead ribozyme and apatamer. <strong>D) </strong>The tertiary structure of natural TPP aptamer. The three-dimensional  segment in blue is TPP, and the yellow part represents the linker between<em> Schistosoma </em>hammerhead ribozyme and  natural TPP aptamer. Nucleotides that bind to TPP are shown in green.</td>
 
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<p class="mainbody">In fact, the natural  aptamer domain of the TPP riboswitch can be exploited to construct very  efficient  that regulate gene expression, demonstrated by Markus Wieland <em>et. al.</em></a><a href="#r301">[3]</a>. To couple the  natural TPP aptamer riboswitch with <em>Schistosoma</em> hammerhead ribozyme, stem III of Schistosoma hammerhead ribozyme and yellow shaded stem of TPP aptamer in <strong>Fig.1-C</strong> were modified to construct  linker between hammerhead ribozyme and aptamer. The resulting artificial  ribozymes functioned with high performance, </a>whose highest fold reached  1000. </p>
 
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<p class="mainbody">Markus Wieland <em>et. al.</em> have created several mutants of  the constructed ribozyme-based TPP-responsive artificial ribozyme switches (TPP  ribozyme)<a href="#r201">[3]</a>. We chose two of the mutants in our project, one of  which can activate downstream gene expression upon adding TPP, numbered 1.20,  and the other would inhibit downstream gene expression when TPP added, numbered  2.5. The secondary structure of TPP ribozyme 1.20 and 2.5 are shown in <strong>Fig.2</strong>. The lower part of the structure  is natural TPP aptamer riboswitch, and the upper part is <em>Schistosoma</em> hammerhead ribozyme. Stem III in green indicates  ribozyme, the pairing  nucleotides of which is the only distinction between TPP ribozyme 1.20 and 2.5.</p>
 
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    <td colspan="3"><p align="left" class="picturemark"><strong>Figure.2</strong><strong> </strong><br />
 
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      Secondary structure of artificial  thiamine pyrophosphate (TPP) ribozymes.<strong> Left:</strong> TPP ribozyme 1.20; <strong>Right:</strong> TPP  ribozyme 2.5. The natural TPP aptamer domain (blue) is fused to stem III of the hammerhead ribozyme. The linker  between aptamer and hammerhead ribozyme is shown in green. Stems are indicated by  roman numerals; rate-enhancing interaction between stem I and stem II are shown as gray lines; the  cleavage site is marked by a red arrow. RBS is shaded in pink and the translation  start code (AUG) is shaded in black. The figure was modified from<a href="#r303"> [3]</a>. </p></td>
 
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<p>The ribosomal binding  site (RBS) of TPP ribozyme locates at the extended stem (shaded red in <strong>Fig.2</strong>). The <em>Schistosoma</em> hammerhead domain in TPP ribozyme could perform  self-cleavage when posed in an appropriate conformation, and upon self-cleavage  the RBS would be released from pairing, thus ribosome could get access to RBS  and initiate translation of the downstream gene. Though similar in secondary  structure, TPP ribozyme 1.20 and 2.5 undergo different mechanisms to regulate  the translation of downstream gene. Upon addition of TPP, the aptamer domain  would bind to TPP; while TPP ribozyme 1.20 would change to a conformation that  is suitable for hammerhead domain to cleave itself, TPP ribozyme 2.5 would undergo  a conformational change that would decelerate the self-cleaving rate of hammerhead  domain. Therefore, upon adding TPP, TPP ribozyme 1.20 would facilitate the  translation of downstream gene, whereas TPP ribozyme 2.5 would decrease the  translation strength of downstream gene (<strong>Fig.3</strong>).</p>
 
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    <td><p align="left" class="picturemark"><strong>Figure.5 </strong>Working curves of  TPP ribozyme 1.20 in different constructs. The activation ratio is </a>fluorescence intensity under  given TPP concentrations compared to that of without TPP. Constructed plasmids were transformed into E. coli DH5a cells and characterized in M9 medium with a TPP concentration gradient of  0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3mM. T7-1.20 and 36-1.20 were  induced by 1mM IPTG. pBAD-1.20 and CI-1.20 were induced by 1mM  arabinose. </p></td>
 
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    <td><p align="left" class="picturemark"><strong>Figure.6 </strong>Working curves of TPP  ribozyme 2.5 in different constructs. The inhibition ratio is fluorescence intensity  under given TPP concentrations compared to that of without TPP. Constructed plasmids were transformed into E. coli  DH5a cells and  characterized in M9 medium with a TPP concentration gradient of 0.001, 0.003,  0.01, 0.03, 0.1, 0.3, 1, 3 mM. T7-2.5 and 36-2.5 were induced by 1mM IPTG. pBAD-2.5 and CI-2.5 were  induced by 1mM arabinose. </p></td>
 
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<p class="mainbody">Additionally, to further  confirm that the rise or drop of the working curves in <strong>Fig.5 </strong>or<strong> Fig.6 </strong>on  different TPP concentrations was indeed the contribution of TPP ribozymes, we constructed  a plasmid as control by substituting TPP ribozyme in pBAD-1.20/ pBAD-2.5 to the  native RBS (AAGGAGAT) of TPP ribozyme, termed TPP-RBS (<strong>Fig.4-E</strong>). Similar characterization was performed, and the result showed  that the fluorescence intensity produced by TPP-RBS fluctuated, yet not  significant enough to show a trend to increase or decrease when TPP  concentration went up, compared with the obvious fluorescence intensity change  produced by pBAD-1.20/ pBAD-2.5 (<strong>Fig.7</strong>).  Therefore, we can reach the conclusion that TPP ribozyme 1.20 and 2.5  functioned modularly to regulate downstream gene’s translation strength upon  different concentrations of TPP. </p>
 
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    <td><p align="left" class="picturemark"><strong>Figure.7 </strong>The fluorescence intensity of TPP-RBS, pBAD-1.20 and  pBAD-2.5 under different TPP concentrations. Ordinate axis indicates the fluorescence  intensity normalized by cell density. Constructed  plasmids were transformed into E. coli DH5a cells and characterized in M9  medium with a TPP concentration gradient of 0, 0.001, 0.003, 0.01, 0.03, 0.1,  0.3, 1, 3 mM, with  induction by 1mM arabinose. </p></td>
 
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<p class="mainbody"><span class="Reference">Reference:</span><a name="r101" id="r101"></a><a name="r102" id="r102"></a><a name="r103" id="r103"></a><a name="r201" id="r201"></a><a name="r202" id="r202"></a><a name="r203" id="r203"></a><a name="r204" id="r204"></a><a name="r301" id="r301"></a><a name="r302" id="r302"></a><a name="r303" id="r303"></a><a name="r304" id="r304"></a></p>
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<p align="left" class="mainbody"><strong>[1]</strong>  Monika Martick and William G.Scott.  (2006). Tertiary Contacts Distant from<br />
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  the Active Site Prime a  Ribozyme for Catalysis. Cell 126, 309-320<br />
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  <strong>[2]  </strong>Wade Winkler, Ali Nahvi Ronald R. Breaker. (2002). Thiamine  Derivatives Bind Messenger RNAs Directly to Regulate Bacterial Gene Expression.  Nature 419, 952-956<br />
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        <th height="27" colspan="2" align="left" class="exist" scope="col">Existing Natural Genetic Rheostats</th>
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  <strong>[3]</strong>  Markus Wieland, Armin Benz, Benedikt Klauser, and Jörg S. Hartig. (2009). Artificial Ribozyme Switches Containing Natural  Riboswitch Aptamer Domains. Angew. Chem. 121, 2753-2756 </p>
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<p class="mainbody"><span class="exist"><a href="#start">[TOP]</a></span></p>
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        <td width="41" height="27" align="left">&nbsp;</td>
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        <td width="447" align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit1">TPP-regulated hammerhead ribozyme</a></td>
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        <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit2">Theophylline-responsive riboswitch</a></td>
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        <td colspan="2" align="left" class="exist"><p>Engineered Genetic Rheostat</p></td>
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        <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit3">Engineered group I intron with a theophylline hammerhead ribozyme</a></td>
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      <td width="447" align="left" class="TPPQ1"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit">TPP</a>:regulated hammerhead ribozyme</td>
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        <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit4">Adenine hammerhead ribozyme obtained from screening</a></td>
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      <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit2">THEOPHYLINE</a>: responsive rboswitch</td>
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      <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit3">RIBOZYME</a>:engineered group I intron with a theolline heammerhead ribozyme</td>
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      <td align="left" class="TPP"><a href="https://2011.igem.org/Team:Peking_R/Project/RNAToolkit4">SELECTION</a>:adenine hammerhead ribozyme obtained from screening</td>
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Overview


In recent years, RNA devices have emerged as powerful tools to regulate gene expression in vivo, and particularly, ligand-responsive riboswitches/ribozymes enable us to manipulate translation strength of specific genes upon different concentrations of ligands. Ligand-responsive riboswitches/ribozymes regulate the translation rate of downstream gene by changing conformations, cleaving or splicing upon external addition of ligand. Compared with transcriptional and post-translational regulation, riboswitches/ribozymes function through allostery of RNA structure, which requires little or no assistance from proteins, so the regulation mechanism is relatively simpler and their functions are more decoupled from native biological activities.

In order to fulfill the goal of establishing an extensible and versatile methodology for softcoding of genetic program, our team reached out to a great extent to search for available ligand-responsive riboswitches/ribozymes that meet our criteria and selected them as our genetic rheostats.

Candidates for genetic rheostats must meet two basic criteria: firstly, they should possess a relatively plain dose-response curve, which would allow for precise translation strength modulation within a wide range of ligand concentration; secondly, ligands they recognize should be genetically and biochemically orthogonal to the host cells, in our case, E.coli cells, as much as possible.

Two candidates emerged as promising genetic rheostats that satisfied our requirements: thiamine pyrophosphate (TPP)-responsive hammerhead ribozymes and theophylline-responsive riboswitches. By altering the upstream promoter and downstream coding sequence of the genetic rheostats, we demonstrated that their performance was independent of sequence context, which proved that our genetic rheostats are modular.

To further extend the repertoire of our genetic rheostats, we created a ribozyme that functions with a different mechanism, which has an extreme low basal level. We substituted the aptamer domain of c-di-GMP group I intron to theophylline-responsive aptamer, thus invented a group I intron that senses theophylline to perform splicing function.

Moreover, we introduced a general method to evolve genetic rheostat that senses a new ligand. By coupling an adenine aptamer with hammerhead ribozyme and randomizing nucleotides in the linker domain, we evolved new genetic rheostat through dual selection, whose self-cleavage could be regulated by adenine.