This project has received funding from the European Union&#39;s Horizon 2020 research and innovation program under grant agreement No. 686647. TEM images were recorded on a Morgagni operated at 80 kV at the EM Facility of the Vienna Biocenter Core Facilities GmbH (VBCF). We would like to thank Tadija Kekic for assistance in the graphical design and Elisa De LIano for designing the wireframe nanostructure.

1. Preparing Polycation Stock Solution
<ol>
	<li>LPEI stock solution
	<ol>
		<li>Add 10 mg of LPEI into a glass beaker containing &#60;10 mL of H2O.</li>
		<li>To completely dissolve LPEI, add HCl (32%) till reaching pH 2.0.</li>
		<li>Adjust the pH to 7.0 by adding NaOH (10 M).</li>
		<li>Adjust the volume to the final concentration of 1 mg/mL.</li>
		<li>Filtrate the LPEI stock solution through a 0.22 &#181;m membrane.</li>
	</ol>
	</li>
	<li>Chitosan stock solution
	<ol>
		<li>Dissolve 16.6 mg of chitosan oligosaccharide lactate (Mw ~ 5 kDa, 60% oligosaccharide composition, deacetylated from chitin by 90%) in 1 mL of acetic acid (10%) aqueous media to prepare a stock solution with a concentration of 10 mg/mL.</li>
	</ol>
	</li>
</ol>
2. Purification of DNA Origami Nanostructures
<ol>
	<li>Mix 100 &#181;L of the DNA origami sample (in 5 mM Tris, 1 mM EDTA, 5 mM NaCl buffer (denoted as TB) including 18 mM MgCl2) with the equivalent volume of 22 mM MgCl2 supplemented TB (100 &#181;L) in a 1.5 mL tube.</li>
	<li>Add 200 &#181;L of purification buffer (15% (w/v) PEG 8,000, 5 mM Tris, 1 mM EDTA and 505 mM NaCl). Mix gently by tube inversion.</li>
	<li>Spin the sample at 16,000 &#215; g at room temperature (r.t.) for 25 min.</li>
	<li>Discard the supernatant carefully and resuspend the pellet in 16 mM MgCl2 supplemented TB buffer. The supernatant and the pellet contain excess staple strands and precipitated DNA origami, respectively.</li>
	<li>Incubate the sample for one day at r.t. at 650 rpm in a thermomixer. This step is for complete resuspension of DNA origami.</li>
</ol>
3. Agarose Gel Electrophoresis (AGE)
<ol>
	<li>Add 1.6 g of agarose and 80 mL of 0.5x TAE buffer (20 mM Tris, 0.5 mM EDTA, 10 mM acetic acid, pH 8) into a glass beaker to prepare a 2% agarose gel. Microwave for 5 min. Swirl the contents of the beaker for 1 min in a water bath. Add 352 &#181;L of MgCl2 (2.5 M) to prepare the gel with 11 mM MgCl2. Pre-stain the gel with 10 &#181;L of DNA gel stain.</li>
	<li>Pour the gel solution into a dry gel box and insert a gel electrophoresis comb. Let the solution solidify at r.t. for 15-30 min.</li>
	<li>Supplement the running buffer (0.5x TAE buffer) with 11 mM MgCl2.</li>
	<li>Mix 10-20 &#181;L of the sample with 20% of 6x loading buffer (30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF) and load it into the agarose gel wells.</li>
	<li>Run the gel at 70 V at r.t. for 2.5- 3 h.</li>
	<li>Visualize the gel with a UV scanner.</li>
</ol>
4. Negative Stain Transmission Electron Microscopy (nsTEM)
<ol>
	<li>Apply 5 &#181;L of the diluted nanostructure or the polyplex on a glow-discharged carbon-coated Cu400 TEM grid. Let it adsorb for ca. 1 min.</li>
	<li>Drain excess liquid from the edge of the grid by a piece of filter paper.</li>
	<li>Apply 5 &#181;L of a 2% uranyl acetate aqueous solution and wait for 1 min.</li>
	<li>Use the filter paper edge to completely drain excess liquid from the edge of the grid.</li>
	<li>Let the grid dry completely before injecting it into the TEM.</li>
</ol>
5. Polyplex Formation
NOTE: Illustrative example for preparing a polyplex comprising DNA origami and chitosan at N/P 0.01-8 ratios.
<ol>
	<li>Calculate the number of amines per polycation using the formula-1 (Table 1).</li>
	<li>Measure the concentration of purified DNA origami using an ultra violet spectrophotometer at 260 nm. Use 2 &#181;L of TB buffer as the blank to calibrate the machine. Calculate the &#34;nmol of phosphate&#34; using formula-2.</li>
	<li>Prepare 15 &#181;L of the DNA origami structure including 1 nmol of phosphate.</li>
	<li>Use formula-3 to calculate the volume of chitosan, which is needed for preparing the polyplex at N/P 0.01-8. For example, to prepare the polyplex at N/P 8, 1.8 &#181;L of chitosan (0.8 mg/mL) is needed (N/P is 8, nmol of phosphate is 1, molecular weight of chitosan is 5,000 g/mol, concentration of chitosan stock solution is 0.8 mg/mL and the number of amines per chitosan is 28). Add 13.2 &#181;L of TB to 1.8 &#181;L of chitosan (0.8 mg/mL). Add 15 &#181;L of chitosan solution (0.8 mg/mL) to 15 &#181;L of the DNA origami from step 5.3 to get a DNA origami-chitosan polyplex with NP 8. The polyplex is formed spontaneously.</li>
	<li>Repeat step 5.4 to prepare polyplexes at different N/P ratios.</li>
	<li>Perform an AGE as described in Step 3 (Figure 1A). Include the naked DNA origami as control.</li>
	<li>Image the polyplex as described in Step 4 (Figure 2). 
	 
	Formula-1) number of amine groups per polycation 
	<img alt="Equation 1" src="/files/ftp_upload/58771/58771eq1.jpg" /> 
	 
	Formula-2) mole of phosphate 
	=<img alt="Equation 2" src="/files/ftp_upload/58771/58771eq2.jpg" /> 
	 
	Formula-3) Volume (&#181;L) of polycations 
	=<img alt="Equation 3" src="/files/ftp_upload/58771/58771eq3.jpg" /></li>
</ol>
6. Decapsulation with Dextran Sulphate
<ol>
	<li>Calculate the number of sulphate groups per dextran sulphate using formula-4 (Table 2).</li>
	<li>Calculate the volume of dextran sulphate needed for the decomplexation of the polyplex at a predefined A/P ratio using formula-5. The A/P charge ratio is the ratio between the sulphate groups of the polyanion (A) to the phosphate groups (P) of the DNA origami. An excess amount of dextran sulphate is needed for an efficient decomplexation of the polyplexes (e.g. 500-1,000). For example, to decomplex the polyplex prepared in section 5 (polyplex contains 1 nmol of phosphate) at A/P 1,000, 3.6 &#181;L of dextran sulphate 40 kDa (50 mg/mL) is needed (A/P is 1,000, nmol of phosphate is 1, the molecular weight of dextran sulphate is 40,000 g/mol, the concentration of dextran sulphate stock solution is 50 mg/mL and number of sulphate is 220). Add 3.6 &#181;L of dextran sulphate (50 mg/mL) to the polyplex from step 5.4 and mix well.</li>
	<li>If working with a chitosan polyplex, add NaOH to facilitate the decomplexation process. Add NaOH for a final concentration of 10 mM. Skip this step for LPEI polyplexes.</li>
	<li>Perform an AGE as described in Step 3 (Figure 1B). Include a naked DNA origami and polyplex as control. 
	 
	Formula-4) number of sulphate groups per polyanion= 
	<img alt="Equation 4" src="/files/ftp_upload/58771/58771eq4.jpg" /> 
	 
	Formula-5) Volume (&#181;L) of dextran sulphate 
	=<img alt="Equation 5" src="/files/ftp_upload/58771/58771eq5.jpg" /></li>
</ol>
7. Stability towards Mg Depletion
<ol>
	<li>Wash 20 &#181;L of a DNA origami or the corresponding polyplex (including 2 nmol phosphate) with 4x 600 &#181;L of Mg-zero buffer (5 mM Tris, 1 mM EDTA and 30 mM NaCl) using the ultrafiltration column (MWCO ~ 3 kDa). Spin each wash at 14,000 &#215; g at r.t. for 3-5 min.</li>
	<li>Collect the remaining solution (80 &#181;L) and incubate at 37 &#176;C for one day at 650 rpm in a thermomixer.</li>
	<li>If testing LPEI polyplex, add 2.5 &#181;L of MgCl2 (500 mM) for a final concentration of 16 mM MgCl2. Then, add 7 &#181;L of dextran sulphate-40 kDa (50 mg/mL) (equivalent to A/P 1,000) to unravel the core DNA nanostructures (decapsulation, see Step 6). 
	NOTE: Skip this step if working with chitosan polyplex or naked DNA origami.</li>
	<li>Follow nsTEM imaging as described in Step 3 (Figure 2).</li>
</ol>
8. DNase I Titration Assay of Naked DNA Origami Nanostructures
<ol>
	<li>Mix 3 &#181;L of naked DNA origami including 1 nmol phosphate with 2 &#181;L of 10x DNase I buffer (100 mM Tris-HCl, 25 mM MgCl2, 5 mM CaCl2, pH 7.6), 1 &#181;L of MgCl2 (250 mM), 12 &#181;L of water in 0.2 mL PCR tubes. Add 2 &#181;L of DNase I (10x). Adjust the stock concentration of the DNase I (10x) to get final DNase I concentrations of 0.25-2 U/mL.</li>
	<li>Incubate the samples at 37 &#176;C for 1-2 h in a thermocycler.</li>
	<li>Immerse the samples in ice and deactivate the nucleolytic activity by adding 2 &#181;L of 500 mM dithiothreitol (DTT) and 2.5 &#181;L of EGTA (33 mM).</li>
	<li>Analyze the samples using an AGE as described in Step 3.</li>
</ol>
9. Stability of Polyplexes towards DNase I
<ol>
	<li>Mix 4 &#181;L of a polyplex (including 1 nmol of phosphate, prepared at different N/P ratios) with 1.5 &#181;L of a DNase I buffer (10x), 8 &#181;L of TB and 1.5 &#181;L of DNase I (100 U/mL) in a 0.2 mL PCR tube.</li>
	<li>Incubate the sample for 24 h at 37 &#176;C in a thermocycler.</li>
	<li>Add 2 &#181;L of proteinase K (20 mg/mL) to digest the DNase&#160;I. Incubate for 30 min at 37 &#176;C in a thermocycler. 
	NOTE: Instead of enzymatic digestion, chemical deactivation of DNase I by adding 1.5 &#181;L of DTT (500 mM) and 2 &#181;L of EGTA (33 mM) can be performed.</li>
	<li>Add 3.6 &#181;L of dextran sulphate-40 kDa (50 mg/mL) (equivalent to A/P 1,000) to unravel the core DNA nanostructures.</li>
	<li>Perform AGE as described in Step 3 (Figure 3A, Figure 3C). Include fresh DNA origami as control for measuring and comparing the band intensities on the gel.</li>
	<li>Excise the band on the AGE and extract the DNA origami by a squeeze freeze extraction column based on the protocol provided by the supplier.</li>
	<li>Follow nsTEM imaging as described in Step 4 (Figure 2).</li>
</ol>
10. Stability towards Fetal Bovine Serum (FBS)
<ol>
	<li>Mix 4 &#181;L of naked DNA origami or its corresponding polyplex (including 1 nmol of phosphate) with 17 &#181;L of TB + 10% FBS in 0.2 mL PCR tubes. Use freshly thawed FBS.</li>
	<li>Incubate the sample at 37 &#730;C in a thermal cycler for 24 h.</li>
	<li>Immerse the sample in an ice bath.</li>
	<li>If testing the polyplex, perform the decapsulation process by adding 3.6 &#181;L of dextran sulphate-40 kDa (50 mg/mL). Additionally, add NaOH if working with chitosan polyplexes (see step 6.3)</li>
	<li>Perform an AGE as described in Step 3. Include the fresh DNA origami as control for measuring and comparing the band intensities on the gel.</li>
	<li>Excise the band on the AGE and extract the DNA origami by a squeeze freeze extraction column.</li>
	<li>Add 2 &#181;L of proteinase K (20 mg/mL) to the extracted DNA origami solution (20 &#181;L) in a 0.2 mL PCR tube. Incubate for 30 min at 37 &#176;C in a thermocycler to digest serum proteins bound to the nanostructures.</li>
	<li>Wash the sample with 5x 500 &#181;L of TB including 16 mM MgCl2 using the ultracentrifugation column (MWCO ~ 100 kDa) to wash proteinase K (MW ~ 28.9 kDa) away. Spin each wash at 14,000 &#215; g at r.t. for 3-5 min.</li>
	<li>Perform nsTEM imaging as described in step 4 (Figure 3). 
	NOTE: The gel extracted sample from step 10.6 might be directly used for nsTEM imaging. However, the imaging could be very challenging due to the attachment of serum proteins to the DNA origami nanostructures. Therefore, proteinase K treatment (steps 10.7-10.9) is recommended to facilitate the imaging process.</li>
</ol>
11. Testing the Addressability of Horseradish Peroxidase Enzyme (HRP)-functionalized DNA Origami upon Coating with Polycations
<ol>
	<li>Purify the self-assembled biotinylated-NR (Biotin-NR) as described in Step 2.</li>
	<li>Incubate 200 &#181;L of purified Biotin-NR (36 nM, in 16 mM supplemented TB buffer) with 200 &#181;L of HRP-conjugated streptavidin (540 nM, in TB buffer). Add 4.8 &#181;L of MgCl2 (500 mM) to a final concentration of 14 mM. Incubate at r.t. for 12 h at 650 rpm in a thermomixer</li>
	<li>Purify HRP-functionalized NR (HRP-NR) by a PEG purification method (as described in Step 2) to remove the unbound HRP enzymes. Resuspend the precipitated HRP-NR in TB including 16 mM MgCl2.</li>
	<li>Mix 6.5 &#181;L of purified HRP-NR (36 nM) with 6.5 &#181;L of polycations of variant concentrations to prepare the polyplex at N/P ratios of 1, 2, 4, 10 and 20 (using formula 3).</li>
	<li>Repeat steps 11.2-11.4 for non-biotinylated NR. As the HRP enzyme may bind non-specifically to the DNA origami, the non-biotinylated DNA origami, which is treated with HRP enzymes and PEG purified (similar to Biotin-NR), will serve as the control in the assay.</li>
	<li>Mix 6.5 &#181;L of distilled-water with 6.5 &#181;L of polycations of variant concentrations corresponding to the defined N/P ratios of 1, 2, 4, 10 and 20. Theses samples serve as the blank group.</li>
	<li>Add the prepared solution in steps 11.4-11.6 (12.5 &#181;L) to a 96-well plate including 72.5 &#181;L of HEPES buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO, pH 7.4) and mix well.</li>
	<li>Add 10 &#181;L of 2,2&#39;-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (15 mM).</li>
	<li>Add 5 &#181;L of H2O2 (12 mM) and mix well by pipetting up and down. H2O2 initiates the reaction.</li>
	<li>Measure the absorbance at 421 nm over 4 h with a multimode plate reader (Figure 4A).</li>
</ol>
12. Testing the Addressability of Hemin-binding Aptamers (HBA)-functionalized DNA Origami upon Coating with Polycations
<ol>
	<li>Purify the HBA functionalized-NR (HBA-NR) as described in Step&#160;2. Resuspend the precipitated HBA-NR in TB supplemented with 6 mM MgCl2 (higher salt concentration leads to the aggregation of HBA-NR).</li>
	<li>Mix 10 &#181;L of HBA-NR (40 nM) with 10 &#181;L of polycations at varying concentration to prepare polyplex of defined N/P ratios of 1, 2, 10 and 20.</li>
	<li>Mix 10 &#181;L of naked-NR (40 nM) or distilled-water with 10 &#181;L of polycations at varying concentrations corresponding to N/P ratios of 1, 2, 10 and 20.</li>
	<li>Add the 20 &#181;L prepared solution from steps 12.2 and 12.3 to the 96-well plate including 60 &#181;L of HEPES buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO, pH 7.4) and mix well. Include at least one blank sample replacing the 20 &#181;L polyplex or polycation solution (from steps 12.2 and 12.3) with water.</li>
	<li>Add 5 &#181;L of hemin (0.04 mM) followed by 10 &#181;L of ATBS (15 mM). Place the plate on an orbital shaker for 5 min.</li>
	<li>Add 5 &#181;L of H2O2 (12 mM) and mix well by pipetting up and down.</li>
	<li>Measure the absorbance at 421 nmover 30 min with the multimode plate reader (Figure 4B).</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+protection+and+release+of+DNA:+tools+to+address+a+bottleneck+of+non-viral+gene+delivery.">Balancing protection and release of DNA: tools to address a bottleneck of non-viral gene delivery.</a> Journal of the Royal Society Interface. 7 (1), 67-82 (2010).</li><li>Li, T., Dong, S., Wang, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplex+aptamers+with+peroxidase-like+DNAzyme+functions:+which+is+the+best+and+how+does+it+work.">G-quadruplex aptamers with peroxidase-like DNAzyme functions: which is the best and how does it work.</a> Chemistry - An Asian Journal. 4 (6), 918-922 (2009).</li><li>Cassinelli, V. 1., Oberleitner, B., Sobotta, J., Nickels, P., Grossi, G., Kempter, S., Frischmuth, T., Liedl, T., Manetto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-Step+Formation+of+"Chain-Armor"-Stabilized+DNA+Nanostructures.">One-Step Formation of "Chain-Armor"-Stabilized DNA Nanostructures.</a> Angewandte Chemie International Edition. 54 (27), 7795-7798 (2015).</li><li>Gerling, T., Kube, M., Kick, B., Dietz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-programmable+covalent+bonding+of+designed+DNA+assemblies.">Sequence-programmable covalent bonding of designed DNA assemblies.</a> Science Advances. , 1157 (2018).</li><li>Xia, T., Kovochich, M., Liong, M., Meng, H., Kabehie, S., George, S., Zink, J. I., Nel, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+Coating+Enhances+the+Cellular+Uptake+of+Mesoporous+Silica+Nanoparticles+and+Allows+Safe+Delivery+of+siRNA+and+DNA+Constructs.">Polyethyleneimine Coating Enhances the Cellular Uptake of Mesoporous Silica Nanoparticles and Allows Safe Delivery of siRNA and DNA Constructs.</a> ACS Nano. 3 (10), 3273-3286 (2009).</li></ol>

The authors declare no conflicts of interest related to this report.

In the self-assembly of DNA origami, the staple strands are typically added in 5-10 excess ratio to the scaffold. These excess staple strands also bind to the polycation and form polyplexes in the monometer range which are further difficult to be separated from DNA origami polyplexes. Hence, a critical step in polyplex formation is to remove the excess staple strands and use well-purified DNA origami nanostructures.
Other methods have been developed for stabilizing DNA nanostructures such as the cyclization of DNA strands via a click reaction26. As alkyne- and azide-modified staple strands are required for the formation of interlocked single-stranded rings, this technique is limited for the stabilization of small nanostructures such a DNA catenane, and it is not readily scalable for larger DNA origami structure such as those used in this protocol. Recently, Gerling et al.27 reported a method for creating covalent cyclobutene pyrimidine dimer (CPD) bonds between neighboring thymidines within DNA nanostructures using ultraviolet irradiation. Although this method is site-selective and scalable, its efficiency in protecting DNA origami is much lower than polycation coating. For example, CPD-stabilized DNA origami objects endure only 0.4 U/mL DNase I for 1 h, while polyplexes were stable in the presence of 10 U/mL DNase I for at least one day.
In brief, gene-therapy inspired chitosan and LPEI coating is a low-cost, one-step and efficient method to address the long-term stability of DNA origami nanostructures in Mg-depleted and nuclease-rich media. The reversibility of the polyplex formation facilitates its application in multi-step diagnostic tests where the protection of DNA origami against nucleolytic degradation or salt-depletion is crucial in one step but may cause problems in other steps such as DNA amplification. Additionally, polycation coating is a potential approach for enhancing the cellular uptake of DNA origami nanostructures for drug-delivery applications28.

DNA is a versatile building block for the programmable self-assembly of nanoscale structures1. The most popular method for creating DNA nanostructures is the DNA origami technique, which is based on the self-assembly of a long circular, single stranded DNA scaffold with the aid of hundreds of shorter shape-determining synthetic staple strands2. Today, the creation of DNA nanostructures in almost any geometry and morphology is readily feasible. DNA nanostructures can be site-specifically functionalized with high precision3,4 and can be programmed to undergo allosteric conformational changes5,6. Hence, using DNA as a building material offers the unique opportunity to create programmable and responsive custom-designed nanostructures for applications in biosensing, diagnostics and drug delivery. However, DNA nanostructures are susceptible to digestion by exo- and endo-nucleases, and lattice-based 3D DNA origami nanostructures generally require high salinity buffers (e.g., 5-20 mM Mg+2) to maintain their integrity7,8.
The rapid degradation of DNA by nucleases present in blood and the extracellular matrix is a major obstacle to the efficient in vivo delivery of genetic products into cells9. To overcome this limitation, in non-viral gene delivery, DNA is mixed with a cationic polymer at a defined N/P charge ratio (ratio of amines in polycation to the phosphates in DNA)10. The complex of DNA with a cationic polymer, known as polyplex, protects DNA from nuclease-mediated degradation and enhances its cellular uptake11. Inspired by gene delivery advancements, oligolysine12, oligolysine-polyethylene glycol (PEG) copolymers13, poly (2-dimethylamino-ethylmethacrylate) (PDMAEMA)-based polymers14, and virus capsid proteins15 have been used for stabilizing DNA origami nanostructures.
Recently, we reported a method for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using the natural cationic polysaccharide chitosan and the synthetic linear polyethyleneimine (LPEI) was reported16. This article is an adaptation of our earlier work and describes the detailed protocol for the preparation of polyplexes, their characterization, testing the stability of naked and protected nanostructures in low-salt and nuclease-rich media and examining the addressability of enzyme- and aptamer-functionalized DNA origami nanostructures upon polycation coating.

<table><tbody><tr><td>10&amp;times; DNase I reaction buffer</td><td>New England Biolab (NEB)</td><td>B0303</td><td></td></tr><tr><td>ABTS (2&amp;prime;-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)</td><td>Alfa Aesar</td><td>J65535</td><td></td></tr><tr><td>Amicon ultracentrifugation columns</td><td>Merck Millipore</td><td>UCF500308, UCF510024</td><td></td></tr><tr><td>Chitosan oligosaccharide lactate (M&lt;sub&gt;n &lt;/sub&gt;~ 4000-6000, &gt; 90% deacetylated, 60%. composition oligosaccharide</td><td>Sigma-Aldrich</td><td>523682</td><td></td></tr><tr><td>Design-specific staple strands</td><td>Integrate DNA Technologies (IDT)</td><td></td><td></td></tr><tr><td>Dextran sulfate sodium salt (Mr ~ 4 kDa)</td><td>Sigma-Aldrich</td><td>75027</td><td></td></tr><tr><td>Dextran sulfate sodium salt (Mr ~ 40 kDa)</td><td>Sigma-Aldrich</td><td>42867</td><td></td></tr><tr><td>DNA gel loading dye (6&amp;times;)</td><td>ThermoFischer sceintific</td><td>R0611</td><td></td></tr><tr><td>DNase I (RNase free)</td><td>New England Biolab (NEB)</td><td>M0303</td><td></td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA) BioUltra, anhydrous, &amp;ge;99% (titration)</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>Freeze&#039;N Squeeze DNA Gel Extraction Spin Columns</td><td>Biorad</td><td>7326165</td><td></td></tr><tr><td>Hemin</td><td>Sigma-Aldrich</td><td>H9039</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hydrogen peroixde slution (32 wt%)</td><td>Sigma-Aldrich</td><td>216763</td><td></td></tr><tr><td>LPEI-25 kDa</td><td>Polysciences, Inc</td><td>23966-1</td><td></td></tr><tr><td>NuPAGE Sample Reducing Agent (500 mM dithiothreitol (DTT))</td><td>ThermoFischer sceintific</td><td>NP0004</td><td></td></tr><tr><td>Polyethylene glycol (PEG, M&lt;sub&gt;W &lt;/sub&gt;~ 8000 Da)</td><td>Carl Roth</td><td>O263.1</td><td></td></tr><tr><td>Polyethylenimine, linear, average Mn 10,000, PDI &amp;le;1.2</td><td>Sigma-Aldrich</td><td>765090</td><td></td></tr><tr><td>Polyethylenimine, linear, average Mn 5,000, PDI &amp;le;1.3</td><td>Sigma-Aldrich</td><td>764582</td><td></td></tr><tr><td>Proteinase K solution (20 mg/ml)</td><td>ThermoFischer sceintific</td><td>AM2548</td><td></td></tr><tr><td>Streptavidin-conjugated HRP</td><td>ThermoFischer sceintific</td><td>N100</td><td></td></tr><tr><td>SYBER safe DNA gel stain</td><td>ThermoFischer sceintific</td><td>S33102</td><td></td></tr></tbody></table>

gene-therapy inspired polycation coating for protection of dna origami nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Three DNA origami nanostructures of different configurations were designed, including a nanorod (NR), a nanobottle (NB) and a wireframe nanostructure (WN) (the 3D models of the nanostructures are illustrated in Figure 2). The NR and the NB were designed based on a square and honeycomb lattice, respectively, using caDNAno17 and the WN was created using the Daedalus software18. A comprehensive protocol for the design and self-assembly of DNA nanostructures has been published already19,20,21,22. The shape-specific staple strands were ordered, self-assembled and further purified based on a protocol described by Stahl et al.23 (step 2).
Polyplexes were characterized by the gel retardation assay and nsTEM imaging. Upon mixing the DNA origami with the polycations, a shift in the naked nanostructure band towards the cathode was observed (Figure 1A). This can be attributed to the counterbalancing of the negative charge of phosphate groups upon binding to polycations and the overall size increase of the complex. Adding an extra amount of polyanions such as dextran sulphate can reverse the polyplex formation. In this regard, dextran sulphate of higher molecular weight (MW ~ 40 kDa) showed to be more efficient for the decomplexation compared to lower molecular weight dextran sulphate (MW ~ 4 kDa) (Figure 1B).
While imaging LPEI polyplexes by uranyl acetate negative stain TEM, it was difficult to image any particles. It was hypothesized that LPEI might hinder uranyl acetate from binding to the phosphate backbone due to tight shielding of DNA origami. Therefore, prior to nsTEM imaging, an excess amount of dextran sulphate was added to LEPI polyplexes. The unraveled DNA origami nanostructures showed no sign of defect nor decomposition. In contrary, chitosan polyplexes were successfully imaged with no need for polyanion treatment (Figure 2).
All naked DNA nanostructures (irrespective of their different configurations) were denatured completely after one day of incubation at 37 &#176;C in Mg-zero buffer, as confirmed by nsTEM imaging. In contrast, nanostructures coated with LPEI or chitosan at N/P&#8805;1 remained intact. Similarly, DNase I titration assays showed the susceptibility of unprotected nanostructures towards enzymatic digestion. While naked nanostructures were completely digested in the presence of 1 U/mL DNase I after 2 h, the LEPI or chitosan encapsulated DNA origami stayed intact in Mg-zero buffer supplemented with 10 U/mL DNase I for a minimum of one day (Figure 2). Higher stability towards nucleolytic digestion was achieved by increasing the N/P ratio of the polyplexes. Additionally, LPEI protects the DNA nanostructures more efficiently compared to chitosan (Figure 3A, Figure 3C), probably due to the higher charge density of LEPI, and thus, higher binding affinity towards the DNA24. No difference was observed while using LPEI of different molecular weight (Figure3B).
The addressability of functional groups in DNA nanostructures after encapsulation in a polymer shell is an important feature. Furthermore, the compatibility of polycation coating with the horseradish peroxidase enzyme (HRP) and hemin-binding aptamers (HBA) functionalized DNA origami was examined. Three biotinylated staple strands were protruded from the surface of the NR and then functionalized with HRP conjugated streptavidin (three enzymes per DNA origami). The catalytically highly active (Kd: 439 nM) HBA aptamer PS2.M (5&#39;-GTGGGTAGGGCGGGTGG-3&#39;) was chosen for these experiments25. Up to 24 staple strands were protruded from the NR surface with a 5-nm length linker (5&#39;-AAAAGAAAAGAAAAA-3&#39;) followed by the PS2.M sequence (24 aptamers per DNA origami). No remarkable hindrance of enzymatic or aptamer activity was observed upon coating HRP- or HBA-functionalized DNA origami with LPEI and chitosan at variant N/P ratios (Figure 4A-B)16. However, the kinetic of HRP enzyme has been dramatically changed after binding to the DNA origami (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58771/58771fig1.jpg" /> 
Figure 1: Representative AGE images of polyplex formation and decapsulation. (A) The electrophoretic mobility shift assay for NB mixed with chitosan at N/P ratios of 0.01-8. Each lane contains 1 nmol NB. The first lane is the reference NB. (B) Decapsulation of polyplexes by polyanionic dextran sulphate (DS). This figure has been modified from a previously published figure16. <a href="https://www.jove.com/files/ftp_upload/58771/58771fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58771/58771fig2.jpg" /> 
Figure 2: Negative stain TEM micrographs of DNA origami and polyplexes. 3D model and nsTEM micrographs of naked DNA origami nanostructures (columns 1 and 2). nsTEM micrographs of LPEI polyplexes (after decapsulation) and chitosan polyplexes (columns 3 and 4). nsTEM images of naked and protected DNA origami (polyplex) subjected to Mg depletion (columns 5, 6 and 7), enzymatic degradation (columns 8 and 9), serum digestion (columns 10 and 11) for one day at 37 &#176;C. LPEI-5 kDa was used in this assay. Naked DNA nanostructures were degraded beyond detection on an AGE in the presence of 1 U/mL DNase I or in TB + 10% FBS after 2 h of incubation at 37 &#176;C. Scale bars: 100 nm. This figure has been modified from a previously published figure16. <a href="https://www.jove.com/files/ftp_upload/58771/58771fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58771/58771fig3.jpg" /> 
Figure 3: Representative results of DNase I protection assays. (A) The DNase I protection assay for polyplexes of WN with LPEI-5 kDa, LPEI-10 kDa and LPEI-25 kDa prepared at N/P ratio of 2, 4, 8 and 10. The samples were subjected to 10 U/mL DNase I for 24 h at 37 &#176;C. The last lane is the control WN. The polyplexes were decapsulated prior to loading into the gel. (B) The normalized mean band intensity for polyplexes of DNA origamis with different LPEI extracted from AGE image-A. Y axis represents normalized mean band intensity. (C) The DNase I protection assay of chitosan-WN polyplexes prepared at N/P ratios of 1, 2, 4, 10 and 20. The samples were subjected to 10 U/mL DNase I for 24 h at 37 &#176;C. Lane 6 is the control polyplex (N/P 20). The last lane is the control WN. All chitosan polyplexes were decapsulated prior loading into the gel. This figure has been modified from a previously published figure16. <a href="https://www.jove.com/files/ftp_upload/58771/58771fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58771/58771fig4.jpg" /> 
Figure 4:&#160;Representative results of colorimetric enzyme- and aptamer-functionalized DNA origami assays. (A) The colorimetric assay of enzyme-functionalized nanostructures (HRP-NR) coated with chitosan 3.7 h after the reaction start. (B) Colorimetric assay of aptamer-functionalized nanostructures (HBA-NR) coated with chitosan, 6 min after the reaction initiation. Y axis represents the normalized absorbance. (C) Monitoring the colorimetric assay of HRP and HRP-NR over time. This figure has been modified from a previously published figure16. <a href="https://www.jove.com/files/ftp_upload/58771/58771fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cationic polymers</td>
			<td>Chitosan</td>
			<td>LPEI-5 kDa</td>
			<td>LPEI-10 kDa</td>
			<td>LPEI-25 kDa</td>
		</tr>
		<tr>
			<td>Molecular weight of polymer (kDa)</td>
			<td>5</td>
			<td>5</td>
			<td>10</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Molecualr weight of monomer (g/mol)</td>
			<td>161</td>
			<td>43</td>
			<td>43</td>
			<td>43</td>
		</tr>
		<tr>
			<td>Number of amine per monomer</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Number of amine per polymer</td>
			<td>28</td>
			<td>116</td>
			<td>232</td>
			<td>581</td>
		</tr>
	</tbody>
</table>
Table 1. Calculating the number of amine groups per polycation.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Molecular weight of dextran sulphate (kDa)</td>
			<td>4</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Molecular weight of monomer (g/mol)</td>
			<td>366</td>
			<td>366</td>
		</tr>
		<tr>
			<td>Number of sulphate per monomer</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Number of sulphate per polymer</td>
			<td>22</td>
			<td>220</td>
		</tr>
	</tbody>
</table>
Table 2. Calculating the number of sulphate groups per polyanion.

Watch this Scientific Journal Video about Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures at JoVE.com

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

gene-therapy-inspired-polycation-coating-for-protection-dna-origami

Research

JoVE Journal

Chemistry

9.0K Views.  AIT Austrian Institute of Technology GmbH. De biologische stabilisatie van DNA-nanostructuren is essentieel voor toekomstige in-vivo toepassingen, zoals gerichte levering van geneesmiddelen. Echter, DNA wordt afgebroken in het lichaam door verschillende enzymen. Hier presenteren we een coatingtechniek die DNA-nanostructuren beschermt tegen enzymatische afbraak.De belangrijkste voordelen zijn dat onze coating bestaat uit niet-toxische verbindingen die al worden gebruikt in levende organismen en dat de functionalisering van het o...

Video: Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Bioengineering

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

Assembly of Gold Nanorods into Chiral Plasmonic Metamolecules Using DNA Origami Templates

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic nanostructures. The high spatial precision of a DNA origami-templated assembly allows controlling the coupling between plasmonic resonances of individual particles and enables tailoring optical properties of the constructed nanostructures. Recently, chiral plasmonic systems attracted a lot of attention due to the strong correlation between the spatial configuration of plasmonic assemblies and their optical responses (e.g., circular dichroism [CD]). In this protocol, we describe the whole workflow for the generation of DNA origami-based chiral assemblies of gold nanorods (AuNRs). The protocol includes a detailed description of the design principles and experimental procedures for the fabrication of DNA origami templates, the synthesis of AuNRs, and the assembly of origami-AuNR structures. In addition, the characterization of structures using transmission electron microscopy (TEM) and CD spectroscopy is included. The described protocol is not limited to chiral configurations and can be adapted for the construction of various plasmonic architectures.

We describe the detailed protocol for the DNA origami-based assembly of gold nanorods into chiral plasmonic metamolecules with strong chiroptical responses. The protocol is not limited to chiral configurations and can be easily adapted for the fabrication of various plasmonic architectures.

The inherent addressability of DNA origami structures makes them ideal templates for the arrangement of metal nanoparticles into complex plasmonic ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

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