OpenWetWare:Getting started

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{{OhioMOD2014}}
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<img src="http://openwetware.org/images/e/ed/OHIOMODLOGO2.png" alt="OhioMOD">
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<h1>Welcome to OhioMOD</h1>
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<p1>OhioMOD is Ohio State's undergraduate biomolecular design team.  The team participates in the BIOMOD international competition each year at Harvard University.  The 2014 project will use a folded DNA structure to control miRNA gene regulation in cancer.</p1>
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<h1>Abstract</h1>
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<p2>MicroRNAs (miRs) are short, non-coding RNAs that regulate gene expression and control essential cellular processes, such as cell cycle progression and apoptosis, through interactions with messenger RNA. Dysregulation of miR expression levels can promote tumorigenesis either by elevating oncogene or silencing tumor suppressor gene levels implicating miRs as an attractive therapeutic target. One therapeutic approach is to decrease levels of miRs that target tumor suppressor genes such as PTEN to ultimately promote apoptosis by complementary base pair antagonism via antisense DNA, but limitations exist in regard to antisense DNA cellular delivery. A previous study demonstrated that DNA nanostructures functionalized with small interfering RNA (siRNA) underwent efficient cellular entry and executed targeted gene suppression suggesting strong potential for DNA nanostructures as a nucleotide delivery vehicle. Here, we report the fabrication of a rod-like
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and a block-like DNA nanostructure incorporated with DNA overhangs capable of binding and sequestering miR-21, a PTEN targeting miR that is overexpressed in a variety of malignancies including chronic lymphocytic leukemia (CLL). Proper assembly was confirmed via gel electrophoresis and transmission electron microscopy. Incorporation and functionality of DNA overhangs complementary for miR-21 was confirmed via fluorescent gel imaging. In addition, DNA nanostructures were shown to enter the endolysosomal pathway as revealed via fluorescent microscopy. Furthermore, miR-21 complementary DNA overhang functionalized structures induced marked decreases in OSU-CLL cell viability relative to scrambled control overhang nanostructures as shown via fluorescent microscopy. Future work will directly investigate expression levels of PTEN mRNA and protein using quantitative real time PCR and immunoblot analysis respectively. Our current findings suggest that the complementary miR-21 DNA overhang functionalized DNA nanostructures effectively sequester miR-21 to induce cytotoxicity in OSU-CLL cells. Our results represent a promising antisense DNA delivery therapeutic approach in a CLL leukemia model with implications to a variety of human malignancies.</p2>
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<h1>Project Video</h1>
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</br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD&action=edit">Edit Main Page</a>
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{{OpenWetWare:Getting started/Introduction}} <!-- PLEASE DO NOT MAKE EDITS TO THIS PAGE! If you would like to make test edits, please refer here: http://openwetware.org/wiki/Sandbox -->
{{OpenWetWare:Getting started/Introduction}} <!-- PLEASE DO NOT MAKE EDITS TO THIS PAGE! If you would like to make test edits, please refer here: http://openwetware.org/wiki/Sandbox -->

Current revision

Welcome to OhioMOD

OhioMOD is Ohio State's undergraduate biomolecular design team. The team participates in the BIOMOD international competition each year at Harvard University. The 2014 project will use a folded DNA structure to control miRNA gene regulation in cancer.

Abstract

MicroRNAs (miRs) are short, non-coding RNAs that regulate gene expression and control essential cellular processes, such as cell cycle progression and apoptosis, through interactions with messenger RNA. Dysregulation of miR expression levels can promote tumorigenesis either by elevating oncogene or silencing tumor suppressor gene levels implicating miRs as an attractive therapeutic target. One therapeutic approach is to decrease levels of miRs that target tumor suppressor genes such as PTEN to ultimately promote apoptosis by complementary base pair antagonism via antisense DNA, but limitations exist in regard to antisense DNA cellular delivery. A previous study demonstrated that DNA nanostructures functionalized with small interfering RNA (siRNA) underwent efficient cellular entry and executed targeted gene suppression suggesting strong potential for DNA nanostructures as a nucleotide delivery vehicle. Here, we report the fabrication of a rod-like and a block-like DNA nanostructure incorporated with DNA overhangs capable of binding and sequestering miR-21, a PTEN targeting miR that is overexpressed in a variety of malignancies including chronic lymphocytic leukemia (CLL). Proper assembly was confirmed via gel electrophoresis and transmission electron microscopy. Incorporation and functionality of DNA overhangs complementary for miR-21 was confirmed via fluorescent gel imaging. In addition, DNA nanostructures were shown to enter the endolysosomal pathway as revealed via fluorescent microscopy. Furthermore, miR-21 complementary DNA overhang functionalized structures induced marked decreases in OSU-CLL cell viability relative to scrambled control overhang nanostructures as shown via fluorescent microscopy. Future work will directly investigate expression levels of PTEN mRNA and protein using quantitative real time PCR and immunoblot analysis respectively. Our current findings suggest that the complementary miR-21 DNA overhang functionalized DNA nanostructures effectively sequester miR-21 to induce cytotoxicity in OSU-CLL cells. Our results represent a promising antisense DNA delivery therapeutic approach in a CLL leukemia model with implications to a variety of human malignancies.

Project Video



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