This work was supported in part by National Science Foundation grant 1739308, NSF CAREER grant 1944130 and by the Air Force Office of Science Research grant number FA9550-18-1-0199. &#947;PNA sequences were a generous gift from Dr. Tumul Srivastava of Trucode Gene Repair, Inc. We would like to thank Dr. Erik Winfree and Dr. Rizal Hariadi for their helpful conversations on DNA Design Toolbox MATLAB code. We would also like to thank Joseph Suhan, Mara Sullivan and the Center for Biological Imaging for their assistance in the collection of TEM data.

1. &#947;PNA sequence design
<ol>
	<li>Download the DNA Design Toolbox22 developed by the Winfree Lab at Caltech23 into the folder containing programming scripts for designing sequences.</li>
	<li>Within that sequence design folder, open a fourth-generation programming language compatible with the file extension &#8220;.m&#8221;, and then add the previously downloaded &#8220;DNAdesign&#8221; folder to the path using the following command: 
	&#62;&#62; addpath DNAdesi...

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M., Lin, M., Winfree, E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigms+for+computational+nucleic+acid+design.">Paradigms for computational nucleic acid design.</a> Nucleic Acids Research. 32 (4), 1392-1403 (2004).</li><li>Sen, A., Nielsen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+stability+of+peptide+nucleic+acid+duplexes+in+the+presence+of+organic+solvents.">On the stability of peptide nucleic acid duplexes in the presence of organic solvents.</a> Nucleic Acids Research. 35, 3367-3374 (2007).</li><li>Yang, Z., Williams, D., Russell, A. 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This article focuses on adapting and improving existing nucleic acid nanotechnology protocols towards organic solvent mixtures. The methods described here focus on modifications and troubleshooting within a defined experimental space of select polar aprotic organic solvents. There is yet unexplored potential for other established nucleic acid nanotechnology protocols to be adapted within this space. This could improve potential applications through integration in other fields such as polymer and peptide synthesis which t...

Successful construction of numerous complex nanostructures1,2,3,4,5,6,7,8,9,10,11,12 in aqueous or substantially hydrated media made using naturally occurring nucleic acids such as DNA1,2,3,4,5,6,7,8,9,10 and RNA11,12 has been shown in previous works. However, naturally occurring nucleic acids undergo duplex helical conformational changes or have reduced thermal stabilities in organic solvent mixtures13,14.
Previously, our lab has reported a method towards the construction of 3-helix nanofibers using gamma-position modified synthetic nucleic acid mimics called gamma-peptide nucleic acids (&#947;PNA)15 (Figure 1A). The need for such a development and potential applications of the synthetic nucleic acid mimic PNA has been discussed within the field16,17. We have shown, through an adaptation of the single-stranded tile (SST) strategy presented for DNA nanostructures18,19,20, that 9 sequentially distinct &#947;PNA oligomers can be designed to form 3-helix nanofibers in select polar aprotic organic solvent mixtures such as DMSO and DMF. The &#947;PNA oligomers were commercially ordered with modifications of (R)-diethylene glycol (mini-PEG) at three &#947;-positions (1, 4 and 8 base-positions) along each 12-base oligomer based on methods published by Sahu et al.21 These gamma-modifications cause the helical pre-organization that is associated with the higher binding affinity and thermal stability of &#947;PNA relative to unmodified PNA.
This article is an adaptation of our reported work in which we investigate the effects of solvent solution and substitution with DNA on the formation of &#947;PNA-based nanostructures15. The aim of this article is to provide detailed descriptions of the design as well as detailed protocols for solvent-adapted methods that were developed for the self-assembly and characterization of &#947;PNA nanofiber. Thus, we first introduce the modular SST strategy, a general platform for nanostructure design using the synthetic nucleic acid mimic PNA.
The helical pitch for PNA duplexes has been reported to be 18 bases per turn in comparison to DNA duplexes, which undergo one turn per 10.5 bases (Figure 1B). Therefore, the domain-length of the demonstrated &#947;PNA SSTs was set at 6 bases to accommodate one third of a full turn or 120&#176; of rotation to enable interaction between three triangularly arrayed helices. Also, unlike previous SST motifs, each SST contains just 2 domains, effectively creating a 1-dimensional ribbon-like structure that wraps to form a three-helix bundle (Figure 1C). Each 12-base &#947;PNA oligomer is gamma modified at the 1, 4 and 8 positions to ensure uniformly spaced distribution of mini-PEG groups across the overall SST motif. Additionally, within the motif, there are two types of oligomers: &#8220;contiguous&#8221; strands that exist on a single helix and helix-spanning &#8220;crossover&#8221; strands (Figure 1D). In addition, oligomers P8 and P6 are labelled with fluorescent Cy3 (green star) and biotin (yellow oval), respectively (Figure 1D), to enable detection of structure formation using fluorescence microscopy. Altogether, the SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers through programmed complementarity of each individual domain to the corresponding domain on a neighboring oligomer (Figure 1E).

<table>
	<tbody>
		<tr>
			<td>&#947;PNA strands/oligomers</td>
			<td>Trucode Gene Repair Inc.</td>
			<td></td>
			<td>Section 2.1</td>
		</tr>
		<tr>
			<td>UV-Vis Spectrophotometer</td>
			<td>Agilent</td>
			<td>Varian Cary 300</td>
			<td>Section 3.1.2</td>
		</tr>
		<tr>
			<td>Quartz cuvettes</td>
			<td>Starna</td>
			<td>29-Q-10</td>
			<td>Section 3.1.1</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio Rad</td>
			<td>C1000 touch</td>
			<td>Section 4.1</td>
		</tr>
		<tr>
			<td>0.2 mL PCR tubes</td>
			<td>VWR</td>
			<td>53509-304</td>
			<td>Section 4.5</td>
		</tr>
		<tr>
			<td>Anhydrous DMF</td>
			<td>VWR</td>
			<td>EM-DX1727-6</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>Anhydrous DMSO</td>
			<td>VWR</td>
			<td>EM-MX1457-6</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>Anhydrous 1,4-Dioxane</td>
			<td>Fisher Scientific</td>
			<td>AC615121000</td>
			<td>Section 4.6</td>
		</tr>
		<tr>
			<td>10X Phosphate Buffered Saline (PBS)</td>
			<td>VWR</td>
			<td>75800-994</td>
			<td>Section 3.1.1</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>89085-399</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Glass cover slips</td>
			<td>VWR</td>
			<td>48382-126</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>2% Collodion in Amyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>9817</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Isoamyl Acetate</td>
			<td>VWR</td>
			<td>200001-180</td>
			<td>Section 5.2</td>
		</tr>
		<tr>
			<td>Biotinylated Bovine Serum Albumin (Biotin-BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A8549</td>
			<td>Section 5.3</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>Section 5.4</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sigma-Aldrich</td>
			<td>189730</td>
			<td>Section 5.5</td>
		</tr>
		<tr>
			<td>Trolox</td>
			<td>Sigma-Aldrich</td>
			<td>238813</td>
			<td>Section 5.7</td>
		</tr>
		<tr>
			<td>Total Internal Reflection Fluorescence microscope</td>
			<td>Nikon</td>
			<td>Nikon Ti2-E</td>
			<td>Section 5.8</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Joel</td>
			<td>JEM 1011</td>
			<td>Section 6.6</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0203-N5AC-PO</td>
			<td>Section 6.3</td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td>Section 6.1</td>
		</tr>
		<tr>
			<td>Formvar, 300 mesh, Copper grids</td>
			<td>Ted Pella Inc.</td>
			<td>1701-F</td>
			<td>Section 6.2</td>
		</tr>
		<tr>
			<td>Formvar-Silicon monoxide Type A, 300 mesh, Copper grids</td>
			<td>Ted Pella Inc.</td>
			<td>1829</td>
			<td>Section 6.2</td>
		</tr>
		<tr>
			<td>DNA oligomers/strands</td>
			<td>IDT</td>
			<td></td>
			<td>Section 7.1</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulphate (SDS)</td>
			<td>VWR</td>
			<td>97064-860</td>
			<td>Section 8.1</td>
		</tr>
	</tbody>
</table>

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.

The protocols discussed in the sections above describe the design of an adapted SST motif from DNA nanofibers for the robust generation of self-assembled nanofibers structures using multiple, distinct &#947;PNA oligomers. This section describes the interpretation of data obtained from the successful recreation of the protocols described.
Following the protocol described in section 5 for TIRF imaging of samples of &#947;PNA oligomers annealed in 75% DMSO: H2O (v/v) most readily provi...

Watch this Scientific Journal Video about Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures at JoVE.com

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

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 ...

This novel protocol describes the use of multiple distinct sequences of the synthetic nucleic acid mimic gamma PNA to form nanostructures in specific organic solvent mixtures. The assays described here show the need to adapt existing nucleic acid nanotechnology protocols towards organic solvents where little to no protocols have been published. Practitioners new to this technique might find it difficult to adapt existing protocols developed for aqueous-rich environments into organic solvent-rich environments because of the propensity of the nanomaterial PNA to aggregate.Begin by preparing the gamma PNA stocks. After obtaining HPLC grade purified gamma PNA strands from a commercial manufacturer, resuspend each strand in deionized water to 300 micromolar concentrations. Store the gamma PNAs at minus 20 degrees Celsius for up to several months.When ready to perform self-assembly, prepare anneal batch samples in 75%DMSO, 75%DMF, and 40%1, 4-dioxane. First, prepare 20 micromolar substocks of each oligomer by aliquoting 0.67 microliters from the main stock and adding deionized water for a final volume of 10 microliters. Add one microliter of each oligomer from the 20 micromolar substocks to a 200 microliter PCR tube, which will come to a total volume of nine microliters for nine oligomers.Add 30 microliters of anhydrous DMSO or DMF with an additional one microliter of deionized water for a final volume of 40 microliters. To prepare the 40%1, 4-dioxane batch, add 16 microliters of 1, 4-dioxane and bring the volume to 40 microliters with deionized water. Anneal the samples for 22.5 hours in a thermal cycler cooling from 90 to 20 degrees Celsius.Typically, melting temperatures obtained for two oligomer and three oligomer gamma PNA subsets lie in the range of 40 to 70 degrees Celsius for different solvent conditions. Program the thermal cycler according to manuscript directions. Once annealing is complete, samples can be stored at four degrees Celsius for 12 to 24 hours before characterization.Make a humidity chamber from an empty pipette tips box by filling the box with approximately five milliliters of water. Prepare a 20-fold dilution of commercially available 2%collodion in amyl acetate with isoamyl acetate solvent to create the nitrocellulose solution. Use tweezers to dip a coverslip into the nitrocellulose solution and allow it to air-dry, then make a flow chamber out of a microscope slide, two double-sided tape strips, and the nitrocellulose-coated coverslip.Prepare the biotin BSA solution by weighing one milligram of biotin BSA and dissolving it in one milliliter of PBS. Pipette 15 microliters of the biotin BSA into the flow channel at an angle and incubate it for two to four minutes at room temperature in the humidified chamber. Blow 15 microliters of the wash buffer, which consists of one milligram of BSA in one milliliter of PBS, into the channel to wash away excess biotin BSA, then passivate the surface by incubating the flow channel for two to four minutes in the humidified chamber.Measure 0.5 milligrams of streptavidin and one milligram of BSA, then dissolve both in one milliliter of PBS. Pipette 15 microliters of the streptavidin solution into the flow channel and place it in the humidified chamber for two to four minutes and wash the channel by flowing 15 microliters of wash buffer. Next, flow 15 microliters of the annealed batch of gamma PNA oligomers in the humidified chamber, then wash the unbound nanostructures by flowing 15 microliters of one millimolar Trolox in the same solvent composition as the nanostructures.Transfer the flow channel to the slide holder and image it with a fluorescence microscope equipped with TIRF imaging, a 60X oil immersion objective, and a 1.5X magnifier. Scan the flow channel at either 60 or 90X magnification while monitoring the Cy3 channel. Prepare 20 and 6%SDS stocks according to manuscript directions, then anneal the gamma PNA oligomers by adding one microliter of each 20 micromolar oligomer to a 200 microliter PCR tube.Add 30 microliters of anhydrous DMSO and one microliter of 6%or 20%SDS to the PCR tubes for a final volume of 40 microliters, which will result in a final SDS concentration of 5.25 or 17.5 millimolar respectively. Anneal the gamma PNAs in the thermocycler as previously described. Characterize gamma PNA nanostructures in the presence of SDS using the TIRF or TEM protocol.TIRF imaging of gamma PNA oligomers annealed in 75%DMSO showed well-organized architectures while annealing in 75%DMF resulted in spicule-shaped or needle-like nanostructures. The 40%1, 4-dioxane condition produced a sparse decoration of filamentous nanostructures. Furthermore, the samples of gamma PNA nanotubes formed in 75%DMSO demonstrated bundling of nanofibers at high magnification or nanoscopic resolutions during TEM imaging.Quantitative analysis of the width of the nanostructures showed a median width of 16.3 nanometers with maximum values beyond 80 nanometers. Isosequential DNA oligomers that replaced contiguous gamma PNAs formed straight filamentous structures whereas replacement of crossover gamma PNAs result in stellate structures. The nanostructures that were replaced with contiguous DNA oligomers had median widths of around 19 nanometers.At high SDS concentrations, gamma PNA nanofibers also adopt highly networked morphologies in comparison to its critical micelle concentrations when viewed with TIRF imaging. TEM imaging indicates that gamma PNA assembly in 5.25 millimolar SDS has the most substantial reductions in structural bundling. When attempting this protocol, it is important to remember to maintain the solvent compositions as mentioned because of the propensity of the nanostructures to aggregate.These methods encourage practitioners to pursue other unexplored avenues of different solvent compositions, surfactants, and other PNA DNA nanostructures.

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4.1K Views.  Carnegie Mellon University. This novel protocol describes the use of multiple distinct sequences of the synthetic nucleic acid mimic gamma PNA to form nanostructures in specific organic solvent mixtures. The assays described here show the need to adapt existing nucleic acid nanotechnology protocols towards organic solvents where little to no protocols have been published. Practitioners new to this technique might find it difficult to adapt existing protocols developed for aqueous-rich environments into organic solvent-r...