Team:ZJU-China/project.htm

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<h2 class="acc_trigger">09 <strong>APPLICATIONS</strong></h2>
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<h2 class="acc_trigger">09 <strong>PERSPECTIVES</strong></h2>
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<h2>1. RNA aptamers take place of fluorescent proteins </h2>
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<h2>1. Riboscaffold and targeted drug delivery therapy</h2>
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<p align="justify">&nbsp;</p>
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<p>This is an extension application of our designed clover series of riboscaffold. Some diseases, such as Cancer, will release some small molecular or change microenvironments beside it thus produce detectable signals. Different from using a biosensor to detect these signals, we utilize our scaffold’s aptamer, accompanying with the production of medicine target the disease. If we change Theophylline aptamer into nidus(disease) molecular aptamer, when riboscaffold bind nidus molecular and change conformation, MS2 aptamer & PP7 aptamer are going to set closer. Enzymes which combining MS2 aptamer & PP7 aptamer and producing drugs are ready to catalyze thus bring out targeting agents. It turns out to be a one-stop agency, once detect the focus of diease, will generate corresponding drug targeting the diease. </p>
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<p align="justify">Some RNA aptamers can bind fluorophores, such as 4-hydroxybenzlidene imidazolinone (HBI), 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI), 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), resembling the fluorophore in GFP, and then these RNA-fluorophore complexes enable to emit different colors of fluorescence comparable in brightness with fluorescent proteins. </p>
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<img src=”https://static.igem.org/mediawiki/igem.org/d/da/ZJU_persp_1.png” width=”600px” / >
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<p align="justify">&nbsp;</p>
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Figure1. Riboscaffold which can detect and treat diseases. </p>
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<p align="justify">These RNA-fluorophore complexes could be used to tag RNAs in living cells to reveal the intracellular dynamics of RNA, including RNA-RNA and RNA-protein interactions.</p>
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<p align="justify">&nbsp;</p>
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<h2>2. Shining Riboscaffold</h2>
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<p align="justify">[Reference: Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, RNA Mimics of Green Fluorescent Protein science, 2011 vol 333, 642-646]</p>
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<p>Paige[1] has reported some RNA aptamers that can bind fluorophores, which are small compounds, and in this way resemble the chemical bonds in GFP, then give out fluorescence. </p>
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<p align="justify">&nbsp;</p>
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<h2>2. kinetic investigation of RNA hybridizations and foldings</h2>
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<p>If we use these aptamer in replace of the theophylline aptamer on our riboscaffold, we can make the riboscaffold shining upon the binding of signal compounds mentioned above. This is a cool method to visualize the states, dynamics and localization of riboscaffold in the living cell. </p>
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<p align="justify">&nbsp;</p>
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<img src=” https://static.igem.org/mediawiki/igem.org/9/95/Zju_persp_2.png” width=”600px” / >
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<p align="justify">By introducing fluorophores like 1-ethynylpyrene into the 2-position of RNA adenosine, through an intermolecular interaction of the pyrene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence spectrum changes.</p>
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<p>Figure2. Aptamers that can shine upon binding. </p>
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<p align="justify">&nbsp;</p>
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<p align="justify">With this fluorescence shift, one can distinguish between single-stranded and double-stranded RNA during thermal denaturation. This behavior could be used for the time resolved investigation of RNA hybridizations and folding by fluorescence spectroscopy.</p>
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<h2>3. LEGO Riboscaffold</h2>
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<p align="justify">&nbsp;</p>
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<p>Riboscaffold has unbelievable ability to extend itself through base pairing with each other, just like LEGO bricks! The assembly of LEGO riboscaffolds can load more enzymes and to a large degree accelerate the reaction or artificially construct a longer pathway with high efficiency. For example, artificial TCA cycle abd artificial EMP are promising results. The following pictures show our wide imagination of the possible structure of LEGO riboscaffolds. </p>
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<p align="justify">[Reference: Josef Wachtveitlb, Joachim W. Engels, ect. RNA as scaffold for pyrene excited complexes, Bioorganic & Medicinal Chemistry 16 (2008) 19-26]</p>
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<p align="justify">&nbsp;</p>
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<p>But how to obtain these LEGO riboscaffolds? Wachtveitlb[2] has reported a fantastic method to detect RNA-RNA interaction by introducing fluorophores like 1-ethynylpyrene into the 2-position of RNA adenosine. When two single-stranded RNAs with this fluorophore base pair with each other, the fluorescence spectrum changes and thus suggesting their interaction. So it is hopeful to find the desired riboscaffolds as LEGO bricks by selecting from the library! </p>
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<h2>3. Medicine & health</h2>
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<img src=” https://static.igem.org/mediawiki/igem.org/b/bc/Zju_persp_4.png” width=”600px” / >
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<p align="justify">&nbsp;</p>
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<p> LEGO bricks.</p>
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<p align="justify">To date, many groups have successfully identifi ed aptamers with a variety of functions, including inhibitory and decoy-like aptamers, regulatable aptamers, multivalent/agonistic aptamers, and aptamers that act as delivery vehicles. Each of these classes of aptamers has potential applications in therapeutics and/or diagnostics.</p>
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<img src=” https://static.igem.org/mediawiki/igem.org/5/56/Zju_persp_5.png” width=”600px” / >
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<p align="justify">&nbsp;</p>
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<p> Long scaffold that has multiple binding sites.</p>
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<p align="justify">Inhibitory aptamers:The most extensively characterized inhibitory aptamer is the RNA aptamer that targets VEGF. This aptamer was approved by the FDA in December 2004, for the treatment of wet age-related macular degeneration (AMD)</p>
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<img src=” https://static.igem.org/mediawiki/igem.org/e/ea/Zju_persp_6.png” width=”600px” / >
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<p align="justify">&nbsp;</p>
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<p> A possible device built by LEGO riboscaffold.</p>
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<p align="justify">Decoy-like aptamers:By mimicking the target sequence of the proteins, aptamers can act as decoys to inhibit binding of transcriptional factors such as HIV-tat, NF-κB, and E2F to their cognate sequences on DNA and thus prevent transcription of target genes and may result in powerful therapeutics for treating many human pathologies</p>
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<img src=” https://static.igem.org/mediawiki/igem.org/8/81/Zju_presp_7.png” width=”600px” / >
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<p align="justify">&nbsp;</p>
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<p> Sheets and tubes constructed by LEGO riboscaffolds in vivo.</p>
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<p align="justify">Multivalent aptamers: A bivalent aptamer targeting HIV has also been described and consists of 2 separate RNA aptamers that bind to 2 distinct stem-loop structures within the HIV 5′UTR: the HIV-1 TAR element and the dimerization initiation site. Similarly, bivalent aptamers targeting thrombin have been engineered as a way to increase the avidity of the aptamer for its target and enhance the anticoagulation effect</p>
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<p align="justify">&nbsp;</p>
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<h2>4. Mimic Long ncRNA</h2>
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<p align="justify">Aptamers as delivery tools: Several groups have reported linking siRNAs to aptamers as a way to specifi cally deliver siRNAs to target cells. Aptamers are also being utilized to deliver toxins, radioisotopes, and chemotherapeutic agents encapsulated in nanoparticles.</p>
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<p>In eukaryote, there are naturally produced long non-coding RNAs that attract more and more attention these days and display intriguing potential to act as scaffolds [3]. And our riboscaffold can mimic them and bring their functions to prokaryote. One of the functions is combining related transcription factors and bring them to promoter, as a result enhance the expression of target gene. That is because ncRNA can binds both DNA and Proteins, and can travel freely between nucleus and cytoplasm, which displays great advantage as a bridge. </p>
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<p align="justify">&nbsp;</p>
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<p align="justify">[Reference: Kristina W. Thiel and Paloma H. Giangrande, Therapeutic Applications of DNA and RNA Aptamers. Oligonucleotides, 2009, Volume 19, Number 3, 209-222]</p>
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<p>Aptamers can be selected in vitro against nearly any target of choice. There are RNA aptamers that can specifically bind some transcriptional regulator. For example, Hunsicker [4] has selected one RNA aptamer that can bind TetR, which usually binds on operator sequence and repress gene expression. So once aptamers mentioned above are designed into a riboscaffold, it can initiate the expression of target genes with higher efficiency. </p>
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<p align="justify">&nbsp;</p>
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<img src=” https://static.igem.org/mediawiki/igem.org/1/15/Zju_persp_3.png” width=”600px” / >
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<h2>4. Regular of gene expression</h2>
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<p>Figure3. Riboscaffold that can bring transcription factors to promoters. </p>
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<p align="justify">&nbsp;</p>
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<p align="justify">Aptamers are small oligonucleic acid molecules that can be selected in vitro against nearly any target of choice. And they often show remarkable binding affinity and specificity, and consequently have a huge potential for application. One of their usages is to play a role in activating gene expression.</p>
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<h2>5. Medicine & Health</h2>
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<p align="justify">&nbsp;</p>
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<p>To date, many groups have successfully identified aptamers with a variety of functions, including inhibitory and decoy-like aptamers, regulatable aptamers, multivalent/agonistic aptamers, and aptamers that act as delivery vehicles [5]. </p>
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<p align="justify">Some RNA aptamers can specifically bind some transcriptional regulator. For example, people have selected one RNA aptamer that can bind TetR, which usually binds on operator sequence and repress gene expression. So once the RNA aptamer binds to the transcriptional regulator, the targeting gene-expression is activated.</p>
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<p align="justify">&nbsp;</p>
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<p>By designing these different aptamers into our RNA scaffold, we can endow our scaffold various potential applications in therapeutics and/or diagnostics. </p>
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<p align="justify">[Reference: Anke Hunsicker, Markus Steber, ect. An RNA Aptamer that Induces Transcription, Chemistry & Biology, 2009,Volume 16, Issue 2, 173–180] </p>
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<p align="justify">&nbsp;</p>
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<p>For instance, designing inhibitory aptamers that targets VEGF into our RNA scaffold can be used to treat the wet age-related macular degeneration and that has been approved by the FDA in December 2004. </p>
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<p>Designing Decoy-like aptamers that can mimic the target sequce of the proteins into our RNA scaffold can be used as decoys to inhibit binding of transcriptional factors such as HIV-tat, NF-κB, and E2F to their cognate sequences on DNA and thus prevent transcription of target genes and may result in powerful therapeutics for treating many human pathologies. </p>
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<p>Designing aptamers behavior as delivery tools into our RNA scaffold can be used to deliver not only some siRNAs to target cells but also toxins, radioisotopes, and chemotherapeutic agents encapsulated in nanoparticles. </p>
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<h2>References:  </h2>
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<p> [1] Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, RNA Mimics of Green Fluorescent Protein science, 2011 vol 333, 642-646. </p>
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<p> [2] Josef Wachtveitlb, Joachim W. Engels, ect. RNA as scaffold for pyrene excited complexes, Bioorganic & Medicinal Chemistry 16 (2008) 19-26. </p>
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<p> [3] Mitchell Guttman, John L. Rinn. Modular regulatory principles of large non-coding RNAs. Nature. 2012 Feb 15;482(7385):339-46. </p>
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<p> [4] Anke Hunsicker, Markus Steber, ect. An RNA Aptamer that Induces Transcription, Chemistry & Biology, 2009,Volume 16, Issue 2, 173–180. </p>
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<p> [5] Kristina W. Thiel and Paloma H. Giangrande, Therapeutic Applications of DNA and RNA Aptamers. Oligonucleotides, 2009, Volume 19, Number 3, 209-222. </p>
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<p> [6] Thodey, K. & Smolke, C.D. Bringing It Together with RNA. Science 333, 412-413 (2011). </p>
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Revision as of 19:00, 26 September 2012

PROJECT

01 ABSTRACT

02 BACKGROUND

03 S0: BASIC RNA SCAFFOLD

04 S1: RIBOSCAFFOLD

05 S2: SCAFFOLD LIBRARY

06 S3: BIOSYNTHESIS OF IAA

07 PARTS

08 RESULTS

09 PERSPECTIVES