Name: Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures (Video) | JoVE Journal
Uploaded: 2020-06-26T14:00:00.000+00:00
Duration: 8 min 15 s
Description: Learn to self-assemble gamma-modified peptide nucleic acids into complex nanostructures using organic solvent mixtures. This protocol enables researchers to adapt nucleic acid nanotechnology for non-aqueous environments.

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5 minutes

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 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>&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

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 γPNA 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 γPNA oligomers annealed in 75% DMSO: H2O (v/v) most readily provides eviden...

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

Research

JoVE Journal

Bioengineering

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.

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

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

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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.

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

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.

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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.

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

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.

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Learn to self-assemble gamma-modified peptide nucleic acids into complex nanostructures using organic solvent mixtures. This protocol enables researchers to adapt nucleic acid nanotechnology for non-aqueous environments.

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

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