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| − | <p>
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| − | <b>AeBlue</b>: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
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| − | activity. After digestion, the interaction between the two strands is too weak to hold the dimer and it
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| − | decays. We can then use the DNA-strand as template to translate the chromoprotein <a href="http://parts.igem.org/Part:BBa_K864401">aeBlue</a>.
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| − | <img src="https://static.igem.org/mediawiki/2017/9/90/T--Munich--Description_aeBlue.svg" width=360>
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| − | <img src="https://static.igem.org/mediawiki/2017/6/64/T--Munich--Description_Intein_Extein.svg" width=360>
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| − | </td>
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| − | <p>
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| − | <b>Intein-Extein</b>: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
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| − | to regulate the protease's diffusion and use it to cleave a TEV tag separating the intein regions of a
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| − | modified chromophore. After the first cleavage, the intein segment excises itself<sup><a class="myLink" href="#ref_13">13</a></sup>, bringing together the
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| − | halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
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| − | read-out.
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| − | <b>Gold nanoparticles</b>: Other than in the other two colorimetric readouts, aeB lue and Intein-Extein, the only protein involved in the gold nanoparticle (AuNP)-readout is Cas13a, like in our RNase Alert readout. This reduces the necessary fine tuning of the biochemical circuit to a minimum, favoring high robustness of the readout. Due to the phenomenon of Localized Surface Plasmon Resonance, AuNPs appear in a distinct color, ranging from intense red to blue, black and colorless. This property depends on particle size, shape, the immediate environment, and -most critical for our purpose- aggregation state<sup><a class="myLink" href="#ref_14">14</a></sup>.
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| − | </p>
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| − | <p> . In our project we use AuNPs with a diameter of roughly 10 nm, giving them a bright red color in solution. Their small size and therefore high surface-to-volume ratio makes them ideal for functionalization with thiolated compounds, forming covalent Au-S bonds. The first step of our concept is to use these properties to functionalize AuNPs with either 5’- or 3’- thiolated DNA and, through addition of linker- RNA which hybridizes with both thiolated DNA strands, form aggregates, changing the color from red to blue. The design of the linker-RNA includes an uracil-rich, single-stranded segment between the DNA-complementary termini, making it prone to Cas13a-mediated promiscuous cleavage. .
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| − | </p>
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| − | <p>
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| − | It has been shown that, for purely DNA-based hybridization, AuNP aggregates can be spotted on filter paper, dried and severed by addition of a nuclease-containing solution, visible through diffusion of red AuNPs on the paper. Thus, the second part of our concept is to spot RNA-linked AuNPs on paper, dry them alongside the Cas13a mixture and detect specific target RNAs and resulting Cas13a activity with a simple change from blue to red.
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| − | </p>
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| − | <img src="https://static.igem.org/mediawiki/2017/b/b3/T--Munich--Description_Goldnanoparticles.svg" width=360>
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| − | <h3>Software</h3>
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| − | <p>
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| − | To help facilitate the design of crRNA, the sequences that give CascAID its specificity, we developed a
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| − | software tool that checks crRNA for unwanted secondary structures. This gives valuable insight on
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| − | whether the sequence is suited to use with Cas13a or whether some modifications are needed.
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| − | Together with Team Delft's software tool which designs the corresponding crRNA based on the target,
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| − | we collaborated to develop a powerful tool that suggests crRNA sequences and checks their usability
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| − | </p>
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| − | </td>
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| − | <h3>References</h3>
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| − | <p>
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| − | <ol style="text-align: left">
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| − | <li id="ref_1">Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).</li>
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| − | <li id="ref_2">Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.</li>
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| − | <li id="ref_3">Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.</li>
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| − | <li id="ref_4">Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.</li>
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| − | <li id="ref_5">Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.</li>
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| − | <li id="ref_6">Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.</li>
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| − | <li id="ref_7">Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.</li>
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| − | <li id="ref_8">Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.</li>
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| − | <li id="ref_9">Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.</li>
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| − | <li id="ref_10">Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.</li>
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| − | <li id="ref_11">Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.</li>
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| − | <li id="ref_12">https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)</li>
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| − | <li id="ref_13"> Anraku, Yasuhiro, Ryuta Mizutani, and Yoshinori Satow. "Protein splicing: its discovery and structural insight into novel chemical mechanisms." IUBMB life 57.8 (2005): 563-574.</li>
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| − | <li id="ref_14">Link, Stephan, and Mostafa A. El-Sayed. "Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles." The Journal of Physical Chemistry B 103.21 (1999): 4212-4217.</li>
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| − | <li id="ref_15">Zhao, W., Ali, M.M., Aguirre, S.D., Brook, M.A., and Li, Y. (2008). "Paper-based bioassays using gold nanoparticle colorimetric probes." Analytical Chemistry 80, 8431–8437.</li>
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| − | </ol>
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| − | </p>
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| − | </td>
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