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>Brief introduction to RNA toolkit</p>
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  <p class="mainbody">In recent years, RNA devices have emerged  as powerful tools to regulate gene expression <em>in vivo</em>, 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.</p>
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  <p class="mainbody">In order to fulfill the goal of establishing  an extensible and versatile platform 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 into our RNA  toolkit. </p>
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  <p class="mainbody">RNA controller candidates 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, <em>E.coli</em> cells, as much as possible. </p>
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  <p class="mainbody">Two candidates emerged as promising RNA  tools 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 RNA controllers, we  demonstrated that their performance was independent from sequence context,  which proved the modularity of these RNA devices.</p>
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  <p class="mainbody">To further extend the range of application  of our RNA toolkit, we created a ribozyme that functions with a different  mechanism, which has an extreme low level of backgrounds. We substituted the  aptamer domain of c-di-GMP group Iintron to theophylline-responsive aptamer,  thus invented a group I intron that senses theophylline to perform splicing  function. </p>
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  <p class="mainbody">Moreover, we introduced a general method to  evolve hammerhead ribozyme that senses a new ligand. By coupling an adenine  aptamer with hammerhead ribozyme and randomizing nucleotides in the linker  domian, we evolved new hammerhead ribozymes through dual selection, whose  self-cleavage could be regulated by adenine. </p>
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   <p class="mainbody">In summary, our project provided new design  principles for rational or semi-rational design of riboswitches/ribozymes. </p>
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        <th colspan="2" align="left" class="exist" scope="col">Existed Natural RNA Controllers</th>
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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/RNAToolkit">TPP</a>:regulated hammerhead ribozyme</td>
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        <td height="28" align="left">&nbsp;</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 colspan="2" align="left" class="exist"><p>Engineered RNA controllers</p></td>
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        <td height="27" align="left">&nbsp;</td>
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        <td align="left" class="TPPQ"><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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      </tr>
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        <td height="33" align="left">&nbsp;</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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      </tr>
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    </table>
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  </div>
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    <table width="673" border="0" cellspacing="0" cellpadding="0">
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      <tr>
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        <th colspan="2" align="left" class="exist" scope="col">Existed Natural RNA Controllers</th>
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      </tr>
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      <tr>
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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</a>:regulated hammerhead ribozyme</td>
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      </tr>
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      <tr>
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        <td height="28" align="left">&nbsp;</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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      </tr>
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      <tr>
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        <td colspan="2" align="left" class="exist"><p>Engineered RNA controllers</p></td>
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      </tr>
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      <tr>
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        <td height="27" align="left">&nbsp;</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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      </tr>
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      <tr>
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        <td height="33" align="left">&nbsp;</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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  <p align="center" class="TITLE2"><strong>High-performance RNA Controllers Regulated By TPP</strong></p>
 
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<p class="mainbody">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 class="mainbody">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>. More recently a  full-length hammerhead ribozyme from <em> mansoni</em> is being more frequently utilized for application. As shown in <strong>Fig.1-A</strong>, this hammerhead ribozyme can  be truncated to a minimal, catalytically active motif consisting of (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>. 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 derivative without the assistance  from protein cofactors <a href="#r201">[2]</a>. These ligand-binding mRNAs actually  possess thiamine or pyrophosphate binding domain, called  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 . <strong>Fig.1-D</strong> shows the tertiary structure  of natural TPP aptamer binding to TPP.</p>
 
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    <td colspan="3"><p align="left"><strong>Figure.1</strong> <br />
 
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        <span class="picturemark">The schematic structures  of  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.</span></p></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 <em>Schistosoma</em> 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 Schistosoma </em></a>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 class="mainbody">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.</p>
 
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      Allosteric mechanism of TPP  ribozymes. Upon self-cleavage the RBS would be released from pairing, thus  ribosome could get access to RBS and initiate translation of the downstream  gene .<strong>A) </strong>Mechanism for TPP  ribozyme 1.20. Upon addition of TPP, TPP ribozyme 1.20 adopts a conformation that  facilitates the self-cleavage of hammerhead domain. </a><strong>B)</strong> Mechanism for TPP ribozyme 2.5. When  TPP added, TPP ribozyme 2.5 would change to a conformation that hinders the  self-cleavage of hammerhead domain. Red: RBS sequence, blue: natural  TPP aptamer, green: linker between aptamer and hammerhead domain, black: Schistosoma  hammerhead ribozyme, red arrow: self-cleavage site. The figure is modified from<a href="#r304"> [3]</a>. </p><p align="left" class="picturemark">&nbsp;</p></td>
 
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<p class="mainbody">We designed several  experiments to demonstrate that TPP ribozymes are truly modular RNA controllers,  independent of sequence context. The plasmids kindly provided by Prof. Hartig’s  lab contain TPP ribozyme 1.20/2.5 with an upstream T7 promoter, and the  downstream coding sequence is a copy of GFP gene, followed by a T7 terminator (<strong>Fig. 4-A</strong>). We termed these two  constructs T7-1.20 and T7-2.5 respectively. To prove that the performance of  TPP ribozymes is not relevant to the downstream coding sequence, we inserted first  36 base pairs of the coding sequence from Part BBa_E0040 ahead of the GFP coding  sequence in the plasmid (<strong>Fig. 4-B</strong>).  The two constructs were termed 36-1.20/36-2.5 respectively. </p>
 
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      Scheme of constructs designed to demonstrate the  modularity of TPP ribozymes. <strong>A)</strong> T7-1.20/T7-2.5  consists of T7 promoter, TPP ribozyme, GFP and T7 terminator with native RBS. <strong> B)</strong> 36-1.20/36-2.5 were constructed by inserting the first 36 base pairs of  BBa_E0040 between TPP ribozyme and GFP. <strong>C)</strong> pBAD-1.20/pBAD-2.5 consists of pBAD promoter, TPP ribozyme, BBa_E0040 and  BBa_B0015 with native RBS (AAGGAGAT). <strong>D</strong><strong>）</strong>CI-1.20/CI-2.5  were constructed by adding the first 36 base pairs of CI ahead of coding  sequence of E0040. <strong>E)</strong> TPP-RBS consists  of pBAD promoter, native RBS, BBa_E0040 and BBa_B0015.</p></td>
 
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<p class="mainbody">After transforming T7-1.20,  T7-2.5, 36-1.20, 36-2.5 into <em>E. coli</em> DH5αcells,  the bacteria were characterized in M9 medium with TPP concentration gradient. The  result turned out that the corresponding working curves were similar, despite  of different downstream coding sequence (<strong>Fig.5  &amp; Fig.6</strong>, grids and curves in black and in red). </p>
 
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<p class="mainbody">To further investigate  whether the performance of TPP ribozymes is influenced by upstream promoter and  downsteam terminator, we constructed two plasmids that contain TPP ribozyme  1.20/2.5 with an upstream pBAD promoter, and the downstream coding sequence is  a GFP gene from Part BBa_E0040, followed by a terminator (Part:BBa_B0015) (<strong>Fig. 4-C</strong>). These two constructs were  named pBAD-1.20 and pBAD-2.5 respectively. Similarly, we characterized these  two constructs, and the working curves were also similar to those of T7-1.20/  T7-2.5 (<strong>Fig.5 &amp; Fig.6</strong>, grids and  curves in black and in blue). Another two plasmids were constructed by  inserting first 36 base pairs of CI gene ahead of GFP coding sequence in  pBAD-1.20/ pBAD-2.5 (<strong>Fig. 4-D</strong>). These  are named CI-1.20 and CI-2.5 respectively. The working curves of these two  constructs overlapped to a large extent with those of other constructs (<strong>Fig.5 &amp; Fig.6</strong>, grids and curves in  green), further demonstrating that the performance of TPP ribozyme 1.20 and 2.5  was independent of their coding sequence context, rendering them truly modular  RNA devices to regulate gene expression. </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. </a>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, mM. T7-1.20 and 36-1.20 were  induced by 1mM IPTG. pBAD-1.20</a> and CI-1.20 were induced by 1mM  arabinose. </p></td>
 
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    <th scope="col"><img src="https://static.igem.org/mediawiki/2011/e/e9/PekingR_Zyy_fig6.png" width="500" height="330" /></th>
 
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    <td>&nbsp;</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 <a name="OLE_LINK8" id="OLE_LINK8"></a><a name="OLE_LINK7" id="OLE_LINK7">m</a>M. 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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    <th scope="col"><img src="https://static.igem.org/mediawiki/2011/4/4f/PekingR_Zyy_fig7.png" width="500" height="330" /></th>
 
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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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  <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">&nbsp;        </p>
 
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<p class="mainbody"><span class="exist"><a href="#start">[TOP]</a></span></p>
 
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      <th colspan="2" align="left" class="exist" scope="col">Existed Natural RNA Controllers</th>
 
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      <td width="41" height="27" align="left">&nbsp;</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 height="28" align="left">&nbsp;</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 colspan="2" align="left" class="exist"><p>Engineered RNA controllers</p></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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Revision as of 23:18, 5 October 2011

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Brief introduction to RNA toolkit

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 platform 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 into our RNA toolkit.

RNA controller candidates 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 RNA tools 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 RNA controllers, we demonstrated that their performance was independent from sequence context, which proved the modularity of these RNA devices.

To further extend the range of application of our RNA toolkit, we created a ribozyme that functions with a different mechanism, which has an extreme low level of backgrounds. We substituted the aptamer domain of c-di-GMP group Iintron 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 hammerhead ribozyme that senses a new ligand. By coupling an adenine aptamer with hammerhead ribozyme and randomizing nucleotides in the linker domian, we evolved new hammerhead ribozymes through dual selection, whose self-cleavage could be regulated by adenine.

In summary, our project provided new design principles for rational or semi-rational design of riboswitches/ribozymes.

Existed Natural RNA Controllers
  TPP:regulated hammerhead ribozyme
  THEOPHYLINE: responsive rboswitch

Engineered RNA controllers
  RIBOZYME:engineered group I intron with a theolline heammerhead ribozyme
  SELECTION:adenine hammerhead ribozyme obtained from screening
Existed Natural RNA Controllers
  TPP:regulated hammerhead ribozyme
  THEOPHYLINE: responsive rboswitch

Engineered RNA controllers
  RIBOZYME:engineered group I intron with a theolline heammerhead ribozyme
  SELECTION:adenine hammerhead ribozyme obtained from screening

 

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