Biomod/2014/OhioMOD/results: Difference between revisions

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</br><img src="http://openwetware.org/images/d/dd/Fig7.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 7: TEM Images of Block O and Branch</font></figcaption>
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<figure>
</br><img src="http://openwetware.org/images/d/d1/Fig8.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 8: TEM Images of Block O and Branch</font></figcaption>
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</br><img src="http://openwetware.org/images/a/a4/Fig9.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 9: TEM Images of Block O and Branch</font></figcaption>
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<figure>
</br><img src="http://openwetware.org/images/e/e9/Fig10.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 10: TEM Images of Block O and Branch</font></figcaption>
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</br><img src="http://openwetware.org/images/0/07/Fig11.png"height="246 " width="576"/>
<figcaption><font size="2">Figure 11: TEM Images of Block O and Branch</font></figcaption>
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<h2>2. miR 21 Sequestering</h2>
<h2>2. miR 21 Sequestering</h2>

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<h1>Project and Results</h1> <h2>Novel microRNA antisense therapeutic delivery using DNA origami nanostructures</h2>


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     <TH COLSPAN="2"><strong>Table of Contents</strong>
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<A HREF="#scroll1">1. Structures</A> </br> <A HREF="#scroll2">2. miR 21 Sequestering</A> </br><A HREF="#scroll3">3. Cellular Uptaking</A> </br> <A HREF="#scroll4">4. PTEN Expression</A> </br><ul id="list2"> <A HREF="#scroll4.1">4.1 Gene Expression</A></ul> <ul id="list2"> <A HREF="#scroll4.2">4.2 Protein Expression</A></ul> <A HREF="#scroll5">5. Cellular Viability</A> <A NAME="scroll1"></A></br> <A HREF="#scroll6">6. Conclusion</A> </br> <A HREF="#scroll7">7. Future Work</A>


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<h2>1. Structures</h2>

<strong>Hypothesis:</strong> <i>Combination of designed staples and scaffold DNA, in an optimized cationic solution will result in the synthesis of nanostructures with specific geometry.</i>

</br></br>Structures were designed to emulate and verify previous reports of ideal nanoparticle size for cellular uptake. It has been demonstrated that rod-shaped nanostructures, with sizes ranging from 20 to 120 nm, are uptaken more effectively than structures with a more cube-like shape[1]. Using this information, we designed two structures at opposite ends of the spectrum. The Block-O is essentially a torus, with outer dimensions of 30x30x24 nm, while the Buckeye Branch is a 90 nm long rod-like shape (Figure 5). Both structures were designed using caDNAno[2] and are able to incorporate ssDNA overhangs complementary to miR-21 (56 overhangs on The Block-O and 42 on the Branch). Three versions of each structure were designed; one with miR-21 complementary overhangs, a control with scrambled-sequence overhangs, and a structure without overhangs. Importantly, the scrambled overhangs were randomized so as to not create any epigenetic effects in the target.


<figure> </br><img src="http://openwetware.org/images/6/6b/1Fig5.png"height="246 " width="576"/> <figcaption><font size="2">Figure 5: Fluorescence microscopy image of Block O structures located outside of cells.</font></figcaption> </figure>


</br>To overcome the electrostatic repulsion of DNA oligomers, the addition of MgCl2 to the folding reaction (a solution containing ssDNA staple strands in a 10 fold molar excess relative to ssDNA scaffold strands, as well as a buffer containing EDTA and Tris) provides divalent cations which facilitating binding interactions. The concentration of these cations has been shown to have a pronounced effect on the yield and quality of folded structures[3]. To optimize this concentration, structures were folded with MgCl2 concentrations ranging from 12mM to 26mM. Subsequent analysis by means of both agarose gel electrophoresis and spectrophotometry showed that a concentration of 20mM MgCl2 provided the highest yields with the lowest concentration of misfolded structures. </br></br>The structures were further validated by direct imaging using Transmission Electron Microscopy (TEM), which showed branch structures with dimensions of 10 nm by 12 nm by 90 nm, and Block O structures with dimensions of 30 nm by 30 nm by 24 nm (Figure 6). This closely matches the designed dimensions. A small degree of global twist is evident in the branch, a result of inter-helical strain induced by the square helix pattern used in the design[4].

<figure> </br><img src="http://openwetware.org/images/9/99/Fig6.png"height="246 " width="576"/> <figcaption><font size="2">Figure 6: TEM Images of Block O and Branch</font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/d/dd/Fig7.png"height="246 " width="576"/> <figcaption><font size="2">Figure 7: TEM Images of Block O and Branch</font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/d/d1/Fig8.png"height="246 " width="576"/> <figcaption><font size="2">Figure 8: TEM Images of Block O and Branch</font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/a/a4/Fig9.png"height="246 " width="576"/> <figcaption><font size="2">Figure 9: TEM Images of Block O and Branch</font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/e/e9/Fig10.png"height="246 " width="576"/> <figcaption><font size="2">Figure 10: TEM Images of Block O and Branch</font></figcaption> </figure>

<figure> </br><img src="http://openwetware.org/images/0/07/Fig11.png"height="246 " width="576"/> <figcaption><font size="2">Figure 11: TEM Images of Block O and Branch</font></figcaption> </figure>

<h2>2. miR 21 Sequestering</h2> <strong>Hypothesis:</strong> <i>Hypothesis: Structures are able to remove miR-21 from solution via complementary base pairing with miR-21 complementary overhangs.</i>

</br></br>In order to demonstrate the ability of the DNA nanostructures to sequester miR-21, structures functionalized with ssDNA overhangs complementary to miR-21 were incubated in solution at 37℃ with fluorescently-tagged miR-21 (Figure 7), purified via agarose gel electrophoresis, and subsequently analyzed by means of bulk fluorescence imaging. Sequestration of miR-21 is evident in sample bands when the fluorescence of the labeled miR-21 is present in the band containing our structures and fluorescence is diminished in the band containing free miR-21. A titration of miR-21 allowed us to determine the ability of the structures to sequester an increasing amount of free miR-21 before becoming saturated (Figure 8). As expected, when the control structure with scrambled overhangs was incubated with fluorescent miR there was no binding observed.


<div id="CellularUptaking"> <h2>3. Cellular Uptaking</h2> </br><strong>Hypothesis:</strong><i> Our structures utilize the endolysosomal pathway to infiltrate cells.</i> </br></br>Once the structures were characterized and functionalized with miR-21 complementary overhangs, the next step was to test whether they would be successfully uptaken into cells. One possible mode of uptake would be through endocytosis, where the structures would be enclosed in an endosome and allowed into the cell. To test this theory, structures labeled with a fluorescent intercalating dye (TOPRO3) were incubated with cells whose lysosomes were labeled with Lysotracker Green. After a 4 hour incubation period, the cells were imaged with a fluorescent microscope to try and observe colocalization between the signals originating from the structures and the lysosomes.

<figure> </br><img src="http://openwetware.org/images/2/25/CellularUptakingfig9.png"height="146 " width="576"/> <figcaption><font size="2">Figure 3.1: Fluorescence microscopy image showing co-localization between structures and endosomes within cells. Top: Branch scrambled overhangs. Bottom: Block O scrambled overhangs.</font></figcaption> </figure> </br>As shown in Figure 9, fluorescence from the Lysotracker (shown in green) and fluorescence from TOPRO3 (shown in red) occupy the same coordinates within a cell, suggesting that the structures and endosomes occupy the same space within the cell. In addition to suggesting a possible mechanism for uptake, the Lysotracker also aids in affirming the position of the structures within the cell. Since these images are two-dimensional, without the Lysotracker, there would be no way to determine whether the signal from the structures originated from within the cell, or from above or below the cell. Co-localizing with Lysotracker affixes the signal’s position in the z-plane as well as the x and y planes.

</br></br>One advantage of generating structures with very different aspect ratios was the ability to test which structures were more easily uptaken by cells. Even though both structures meet the ideal dimensional parameters for cell uptake[22], the different geometries of the elongated branch versus the compact Block O may have an impact on cell uptake efficiency. While microscopy images do not necessarily provide quantitative data on the uptaking efficiency of structures, some preliminary assessments may be made nonetheless. Firstly, more instances of co-localization could be observed in the presence of branch structures relative to Block O structures. Perhaps more intriguingly, in the presence of branch, all instances of fluorescence in the 640 nm channel occurred either within viable cells or within dead cell debris. However, a few instances of fluorescence were found outside of cells in the presence of Block O, as shown in Figure 10.

</br></br>Figure 10 </br>The fluorescence signal, with an intensity comparable to the intensity of the TOPRO3 dye, originated at the very edge of the cell, on the cell membrane. This may suggest that while branch structures are quickly uptaken once they come in contact with cells, the Block O structures experience a delay in uptake, and thus tend to congregate outside the cell. While this introduces an interesting possibility in terms of the mechanism of uptake, more experiments must be conducted both using qualitative methods such as fluorescent imaging, and quantitative methods such as qPCR to detect intracellular levels of DNA nanostructures to form a conclusion on this matter. </div><!--end of cellular uptake-->


<h2>4. PTEN Expression</h2> <h3>4.1 Gene Expression</h3> <h3>4.2 Protein Expression</h3> <h2>5. Cellular Viability</h2>


<figure> </br><img src="http://openwetware.org/images/4/4b/5fig1.png"height="246 " width="476"/> <figcaption><font size="2"></font></figcaption> </figure> <figure> </br><img src="http://openwetware.org/images/4/4f/5fig2.png"height="246 " width="476"/> <figcaption><font size="2"></font></figcaption> </figure>

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<h2>6. Conclusion</h2> <h2>7. Future Work</h2> <br><a href="http://openwetware.org/index.php?title=Biomod/2014/OhioMOD/results&action=edit">Edit Project and Results</a><br><br> </div>


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