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 DNAdesign</li>
	<li>Subsequently run the following script named &#8220;PNA3nanofiber.m&#8221; (see Supplementary Figure 1) using the following command: 
	&#62;&#62; PNA3nanofiber</li>
	<li>Run this script to create variables called &#8220;thisSeqs&#8221; and &#8220;thisScore.&#8221; The variable &#8220;thisSeqs&#8221; contains the sequences of the designed oligomers, and &#8220;thisScore&#8221; is the penalty score.</li>
	<li>Run the script multiple times to obtain the most minimal score. A sample of 20 such runs are shown in Table 1.</li>
	<li>Manually confirm the desired Watson-Crick complementarity of each domain of the generated sequences for the specified SST structural motif. Sequence specifications for the 3-helix nanofiber structure are shown in Table 2.</li>
	<li>Verify the following for generated sequences.
	<ol>
		<li>Avoid using four consecutive C and G bases.</li>
		<li>Manually select specific sequences for N-terminal functionalization with fluorescent dye and biotin molecules to enable fluorescent microscopy studies.</li>
		<li>Include a minimum of 3 mini-PEG gamma-modifications on the backbone to enable pre-organized helical conformation in the single-stranded oligomers as described by Sahu et al.21</li>
	</ol>
	</li>
</ol>
2. Preparation of &#947;PNA stock strands
<ol>
	<li>Obtain &#947;PNA component strands of specified sequences at 50 nmol scale synthesis with high performance liquid chromatography (HPLC)purification from a commercial manufacturer.</li>
	<li>Resuspend each strand in deionized water to 300 &#181;M concentrations. Store the resuspended strands in -20 &#176;C freezer up to several months until needed for experimentation.</li>
</ol>
3. Melting curve studies of &#947;PNA oligomer subsets
<ol>
	<li>Obtain melting temperature ranges for different combinations of complementary 2-oligomer and 3-oligomer subsets by running a melt curve study in aqueous buffers such as 1x phosphate buffered saline (PBS) or preferred polar organic solvent such as Dimethylformamide (DMF) or Dimethyl sulfoxide (DMSO) (see Figure 2). 
	NOTE: Thermal melting curves at &#62;50% of DMF start to lose the upper baseline and show severe disturbances partly because of high absorbance of DMF at the wavelength required for the experiments. This is a noted phenomenon in the literature24. It is however possible to obtain the melting curves at 5 &#181;M per strand concentrations in these solvent conditions.
	<ol>
		<li>Aliquot 16.7 &#181;L from the 300 &#181;M stock of each oligomer and make the final volume to a 1000 &#181;L either in 1x PBS or 100% (v/v) DMSO or 100% (v/v) DMF effectively obtaining 5 &#181;M final concentration per oligomer. Transfer this oligomer subset mixture to a 1 cm optical path, quartz cuvette.</li>
		<li>Perform variable temperature UV-Vis experiments in a spectrophotometer equipped with a programmable temperature block.</li>
		<li>Collect data points for the melting curves over a temperature range of 15&#8210;90 &#176;C for both cooling (annealing) and heating (melting) cycles at a rate of 0.5&#8210;1 &#176;C/min. Keep the samples for 10 min at 90 &#176;C before cooling and at 15 &#176;C before heating. Determine the melting temperature (Tm) from the peak of the first derivative of the heating curve.</li>
		<li>Verify that melting temperature ranges for 2-oligomer subsets are above 35 &#176;C in all solvent cases. Additionally, verify that the corresponding 3-oligomer subsets that contain an additional strand to their equivalent 2-oligomer subset shows a considerable increase in Tm due to increased co-operativity. 
		NOTE: This would verify that a single pot self-assembly of multiple oligomers can co-operatively fold into the desired nanostructure with reasonable thermal stability.</li>
	</ol>
	</li>
</ol>
4. Self-assembly protocol for multiple distinct &#947;PNA oligomers
NOTE: To devise a self-assembly thermal ramp protocol for &#947;PNA nanostructures, slow-ramp annealing is desirable.
<ol>
	<li>In the case of oligomer sequences generated, anneal the samples for 22.5 h in a thermal cycler cooling from 90 to 20 &#176;C. Typically, melting temperature obtained for 2-oligomer and 3-oligomer &#947;PNA subsets lie in ranges of 40&#8210;70 &#176;C for different solvent conditions.</li>
	<li>Program the thermal cycler as follows: hold at 90 &#176;C for 5 min, ramp down from 90 to 70 &#176;C at a constant rate of 0.1 &#176;C/min, ramp down from 70 to 40 &#176;C at a rate of 0.1 &#176;C/3 min, ramp down from 40 to 20 &#176;C at a rate of 0.1 &#176;C/min and hold at 4 &#176;C (see Table 3). Samples can be stored in 4 &#176;C for 12&#8210;24 h before characterization. 
	NOTE: While 2- and 3- oligomer subsets of our nanofiber system can form in 1x PBS, the full micron-scale nanofibers aggregate in 1x PBS. Therefore, solvent conditions should be optimized based on the scale and size of structure being formed as well as the type and density of gamma modifications.</li>
	<li>For micron scale long 3-helix nanofibers, prepare anneal batch samples in 75% DMSO: H2O (v/v), 75% DMF: H2O (v/v), 40% 1,4-Dioxane: H2O (v/v) based on solvent optimization studies15.</li>
	<li>Prepare anneal batches as follows (see Table 4). First, prepare 10 &#181;L sub-stocks at 20 &#181;M concentrations from the 300 &#181;M main stocks for each oligomer by aliquoting 0.67 &#181;L from the main stock and making the volume to 10 &#181;l using deionized water.</li>
	<li>Aliquot 1 &#181;L from the 20 &#181;M sub-stocks for each oligomer and add it to a 200 &#181;L PCR tube. This accounts for a total volume of 9 &#181;L for 9 oligomers.</li>
	<li>Add either 30 &#181;L of anhydrous DMSO/DMF for the 75% DMSO and 75% DMF cases with an additional 1 &#181;L of deionized water to make a final volume of 40 &#181;L with each oligomer at 500 nM final concentrations. Add 16 &#181;L of 1,4-Dioxane and make the volume with deionized water to 40 &#181;L for the 40% Dioxane solvent condition.</li>
	<li>Load the anneal batches on to the thermal cycler and anneal using the protocol mentioned in step 4.2.</li>
</ol>
5. Total internal reflection fluorescence (TIRF) microscopy imaging
<ol>
	<li>Prepare a humidity chamber from an empty pipette tips box. Fill the box with approximately 5 mL of water to prevent drying of the sample flow channels described as follows (see Figure 3).</li>
	<li>Prepare flow chamber with a microscope slide, 2 double-sided tape strips, and nitrocellulose-coated coverslip. To coat the coverslip with nitrocellulose, dip the coverslip in a beaker containing 0.1% collodion in amyl acetate and air-dry.
	<ol>
		<li>Prepare the nitrocellulose solution by making a 20-fold dilution from commercially available 2% collodion in amyl acetate with isoamyl acetate solvent.</li>
	</ol>
	</li>
	<li>Prepare biotinylated bovine serum albumin (Biotin-BSA) solution by weighing 1 mg of Biotin-BSA and dissolving it in 1 mL of 1x PBS. Flow 15 &#956;L of 1 mg/mL Biotin-BSA in 1x PBS buffer by placing the flow channel at an angle. Incubate the flow channel for 2&#8211;4 min at room temperature in a humidified chamber.</li>
	<li>Prepare the wash buffer by dissolving 1 mg of BSA in 1 mL of 1x PBS. Wash the excess Biotin-BSA by flowing 15 &#956;L of the wash buffer. To passivate the surface, incubate the flow channel for 2&#8211;4 mins at room temperature in a humidified chamber.</li>
	<li>Prepare the streptavidin solution by measuring 0.5 mg of streptavidin and 1 mg of BSA and then dissolving using 1 mL of 1x PBS. Flow 15 &#956;L of 0.5 mg/mL streptavidin in 1x PBS containing 1 mg/mL BSA. Incubate the flow channel for 2&#8211;4 min at room temperature in a humidified chamber. Flow 15 &#956;L of the wash buffer to wash away unbound Streptavidin.</li>
	<li>Flow 15 &#956;L of previously annealed batch of &#947;PNA oligomers at 500 nM concentration per strand in either 75% DMSO, 75% DMF or 40% 1,4-Dioxane condition. Incubate the flow channel for 2&#8211;4 mins at room temperature in a humidified chamber.</li>
	<li>Prepare 1 mM Trolox in preferred solvent conditions by diluting 10-fold from a 10 mM stock of Trolox in DMSO (measure 2.5 mg of Trolox and dissolve in 1 mL of DMSO). Wash the unbound nanostructures in the flow channel with 15 &#181;L of 1 mM Trolox having the same solvent composition as the nanostructures. 
	NOTE: The &#62;50% DMSO content in the flow channel would produce minor signal disturbances due to the different index of refraction of the solvent condition during TIRF which is typically calibrated for coverslip-water TIRF angles. This can be rectified by washing with 1 mM Trolox made by diluting the 10 mM Trolox 10-fold using deionized water. This technique provides clearer images but is only useful for assessing whether formation occurred, however, because it produces two-phase micro-bubbles in the flow channel. As a result, nanostructures might be visible at the interface of the two-phases as shown in Figure 4D.</li>
	<li>Transfer the flow channel on to the slide holder and image using a fluorescence microscope equipped with TIRF imaging using a 60x oil-immersion objective and a 1.5x magnifier. Scan the flow channel at either 60x or 90x magnification by monitoring the Cy3 channel (see Figure 4).</li>
</ol>
6. Transmission electron microscopy (TEM) imaging
<ol>
	<li>Weigh 0.5 g of uranyl acetate in 50 mL of distilled water to prepare a 1% aqueous uranyl acetate stain solution. Filter the 1% aqueous uranyl acetate stain solution using a 0.2 &#956;m filter attached to a syringe. 
	NOTE: Alternatively, 2% aqueous uranyl acetate could be purchased from a commercial manufacturer.</li>
	<li>Purchase commercially available formvar support layer coated Copper grids with 300-mesh size. 
	NOTE: It is important to note that the formvar support layer could be dissolved away by solvents like DMSO beyond 2 min. For longer sample incubation, commercially available formvar stabilized with silicon monoxide on copper grids are available which allow the grid to be more hydrophilic than carbon-coated grids and can withstand vigorous sample conditions and electron beam as shown in Figure 5C.</li>
	<li>Pipette 4 &#956;L of sample onto the grid for 15 s. Use a piece of filter paper to wick off the sample by bringing the filter paper in contact with the grid from the side.</li>
	<li>Immediately add 4 &#956;L of the stain solution onto the grid for 5 s.</li>
	<li>Wick off the stain as before and hold the filter paper against the grid for 1&#8210;2 min to make sure the grid is dry. Samples should be typically imaged within 1&#8210;2 h after staining. Alternatively, if the grid is ensured to be completely dry, grids can be stored in a TEM grid storage box for up to 3 days before imaging.</li>
	<li>Transfer the grid to a TEM specimen holder and image using a transmission electron microscope operated at 80 kV with magnification ranging from 10 K to 150K (see Figure 5).</li>
</ol>
7. Different morphologies for &#947;PNA-DNA hybrids based on selective replacement with DNA
<ol>
	<li>Obtain DNA oligomers of specified sequences (see Table 5) from commercial oligonucleotide manufacturers synthesized at 25 nmol scale using standard desalting. Resuspend these DNA sequences using RNase-free deionized water at 20 &#181;M stock concentrations.</li>
	<li>For contiguous &#947;PNA strand replacements with DNA, sequences D3, D5 and D9 can replace strands p3, p5 and p9. Similarly, for crossover &#947;PNA strand replacements with DNA, sequences D1, D4 and D7 can replace strands p1, p4 and p7.</li>
	<li>Aliquot 1 &#181;L from the 20 &#181;M sub-stocks for each &#947;PNA or DNA oligomer and add it to a 200 &#181;L PCR tube as in step 2.7. Add 30 &#181;L of anhydrous DMSO and 1 &#181;L of RNase-free deionized water to make the final volume to 40 &#181;L (see Table 6).</li>
	<li>Load the anneal batches on to the thermal cycler and anneal using the protocol mentioned in step 4.2.</li>
	<li>Characterize &#947;PNA-DNA hybrid nanostructures using the TIRF protocol following steps in section 5 or using TEM imaging protocol mentioned in section 6 (see Figure 6).</li>
</ol>
8. Different morphologies for &#947;PNA nanofibers in varying concentrations of SDS
<ol>
	<li>Prepare a 20% (wt/v) SDS main stock by measuring 20 mg of SDS and dissolving it in 100 &#181;L of deionized water.</li>
	<li>Prepare a 6% (wt/v) SDS sub-stock by aliquoting 3 &#181;L from the 20% SDS stock and making the volume to 10 &#181;L using deionized water.</li>
	<li>Anneal the &#947;PNA oligomers in final concentrations of 5.25 mM and 17.5 mM SDS as follows. Aliquot 1 &#181;L from the 20 &#181;M sub-stocks for each &#947;PNA oligomer and add it to a 200 &#181;L PCR tube as in step 2.7. Add 30 &#181;L of anhydrous DMSO and 1 &#181;L of 6% and 20% SDS to make the final volume to 40 &#181;L to achieve SDS final concentrations of 5.25 mM and 17.5 mM, respectively (see Table 7).</li>
	<li>Load the anneal batches of varying SDS concentrations on to the thermal cycler and anneal using the protocol mentioned in step 4.2.</li>
	<li>Characterize &#947;PNA nanostructures in the presence of SDS using the TIRF protocol following steps in section 5 or using TEM imaging protocol mentioned in section 6 (see Figure 7).</li>
</ol>

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The authors declare no competing financial interests.

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 typically are performed in similar organic solvents25,26. Additionally, we focus here on critical steps to be observed while practicing the above-mentioned protocols.
During the preparation of sample for self-assembly, it is important to keep the volume percentages of water at 25% (v/v) for DMSO and DMF conditions. Accordingly, it is important to recognize that the organic solvent used for self-assembly should be from anhydrous stocks as well. The nanofiber structures are hydrophobic and aggregate in increased water content.
Unlike scaffolded DNA origami approaches that can create structures of discrete lengths, the current SST motif design for &#947;PNA nanofibers and other previously established DNA SST nanotubes does not yield structures of discrete lengths. Nanotubes polymerize achieving a range of multi-micron lengths (up to 11 &#181;m). For the same reason, yield associated with structure formation cannot be quantified.
However, due to the uncharged peptide backbone of &#947;PNA, there are no dependencies on counter ion balance in relation to structure formation. Imaging buffers, therefore, do not need to include cations like Mg2+ typically required for the stabilization of nanostructures made from naturally occurring nucleic acids.
Lastly, &#947;PNA nanostructure bundling and aggregation in different solvent conditions are also dependent on the individual oligomer concentrations introduced during self-assembly. Concentrations ranging from 1 &#181;M and higher of individual oligomers increase the propensity for bundling and aggregation and affect clear imaging of nanostructures either during TIRF or TEM imaging.

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>&amp;gamma;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.

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 ...

self-assembly-gamma-modified-peptide-nucleic-acids-into-complex

Nanyang Technological University

Research

JoVE Journal

Bioengineering

4.1K Views.  Carnegie Mellon University. Dit nieuwe protocol beschrijft het gebruik van meerdere verschillende sequenties van het synthetische nucleïnezuur bootsen gamma PNA na om nanostructuren te vormen in specifieke organische oplosmiddelmengsels. De hier beschreven tests tonen de noodzaak aan om bestaande nucleïnezuurnanotechnologieprotocollen aan te passen aan organische oplosmiddelen waar weinig tot geen protocollen zijn gepubliceerd. Beoefenaars die nieuw zijn voor deze techniek kunnen het moeilijk vinden om bestaande proto...

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

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Chemistry

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

Biochemistry

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

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

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