LM, LDM, and DT acknowledge support from the European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG No 851667 - NANOCELL). LDM acknowledges support from a Royal Society Research Grant for Research Fellows (RGF/R1/180043) and support from a Royal Society University Research Fellowship (UF160152, URF/R/221009).

NOTE: The protocol is divided into three sections. Section 1 describes the prerequisite steps, including the preparation of DNA oligonucleotides and glass capillaries. Section 2 describes the preparation of C-star condensates of various designs, including one- and two-component designs, and their extraction from the glass capillaries. Section 3 describes the use of one-component RNA templating C-star condensates for the synthesis of an RNA aptamer. The user must follow good lab practice throughout, ensure that all necessary risk assessments and mitigations are in place, and wear appropriate personal protective equipment (PPE), including gloves, safety glasses, and a lab coat. The cleaning of glass capillary tubes requires their sonication, first in a surfactant solution and second in isopropanol or ethanol. The extraction of C-star condensates from capillary tubes requires the use of a diamond scribing pen to score and snap the glass, with an associated risk of injury from glass fragments. Key materials, equipment, and reagents used are listed in the Table of Materials. Most non-functionalized oligonucleotides are purified by the supplier using standard desalting, with the exception of the &#34;extended Core 1&#34; and &#34;Aptamer template&#34; strands, which are ordered with polyacrylamide gel electrophoresis (PAGE) purification. Cholesterol-modified oligonucleotides are purified by the supplier using reverse-phase high-performance liquid chromatography (HPLC).
1. Prerequisites
NOTE: The following solutions should be prepared in ultrapure (Type I) water and filtered using 0.22 &#181;m syringe filters: Tris-EDTA (TE) buffer, comprising 10 mM Tris, 1 mM EDTA, at pH ~8.0; TE buffer supplemented with 2 M NaCl; and TE buffer supplemented with 0.3 M NaCl. Buffer solutions should be used within 2 weeks of preparation and stored at 4 &#176;C when not in use. In addition, a 1 vol% solution of alkaline optical detergent in ultrapure water will be used for cleaning the glass capillaries.
<ol>
	<li>Preparation of DNA oligonucleotides from lyophilized state
	<ol>
		<li>Briefly centrifuge (2000 x g for 10-30 s) the tubes of lyophilized DNA oligonucleotides to ensure pellets of lyophilized DNA collect at the bottom. 
		NOTE: The centrifugation is done at room temperature (RT) for 10-30 s. The mini centrifuge used here defaults to 2000 x g (6000 rpm).</li>
		<li>Add the appropriate amount of TE buffer to reconstitute each oligonucleotide at 100 &#181;M. Vortex thoroughly to ensure complete dissolution, then spin down tubes to collect all liquid as per step 1.1.1.</li>
		<li>For each stock solution, measure absorbance at 260 nm and calculate the final concentration using the extinction coefficient of the oligonucleotide sequence under test. 
		NOTE: The extinction coefficient for a given oligonucleotide sequence is usually found on the supplier&#39;s Data Sheet but can also be calculated with online tools using the nearest neighbor model35 or tabulated values of the extinction coefficient of individual nucleotides36.</li>
		<li>Store the rehydrated oligonucleotides at 4 &#176;C for short periods of time (up to 1 week) or at -20 &#176;C for longer-term storage (up to 6 months).</li>
	</ol>
	</li>
	<li>Cleaning of glass capillary tubes
	<ol>
		<li>Sonicate in 1% alkaline optical detergent for optical components in deionized water.
		<ol>
			<li>Take the required number of glass capillary tubes to be cleaned and place them into a tall, narrow container (e.g., a beaker or a 15/50 mL centrifuge tube) such that the capillaries do not lie flat on the base of the container.</li>
			<li>Add the detergent to the container, filling it just above the level of the capillary tubes. Ensure there are no trapped air bubbles inside the capillary tubes - tap to remove, if necessary. Loosely cover the container (e.g., in tin foil or using the centrifuge tube lid, ensuring it is not tightly sealed).</li>
			<li>Set a sonicating water bath to 40 &#176;C, place the covered container upright into the bath, and sonicate for 30 min. Ensure the foil, if used, does not come into contact with the water in the sonicating bath. 
			NOTE: The sonicating bath used in this study defaults to a sonication frequency of 60 Hz and a sonication power of 150 W. 
			CAUTION: Never add or remove items from a sonicating bath while running; always pause the protocol first.</li>
			<li>Once sonication is complete, turn off the heating element of the sonicating water bath, then thoroughly rinse the capillaries in the container with deionized or ultrapure water - a minimum of five times, discarding the rinsing water each time.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sonicate in isopropanol or ethanol.
	<ol>
		<li>To the glass capillary tubes in the cleaning container, add isopropanol or ethanol (minimum 70%), filling to just above the level of the capillary tubes. As in step 1.2.1.2, ensure there are no trapped air bubbles in the capillary tubes and loosely cover the container.</li>
		<li>Place the container upright into the sonicating bath and sonicate for 15-30 min. 
		CAUTION: Isopropanol and ethanol are flammable, with flash points below RT. Minimize the volumes of solvents used during cleaning by choosing a container of appropriate size, ensure that the containers are loosely covered, and do not leave the sonicating bath unattended during this stage.</li>
		<li>Once sonication is complete, appropriately dispose of the isopropanol or ethanol and dry the capillaries under nitrogen, handling them with a lint-free tissue.</li>
	</ol>
	</li>
</ol>
2. Preparation and extraction of C-star condensates (Figure 2)
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66738/66738fig02.jpg" /> 
Figure 2: Loading C-star mixtures and extracting condensates from glass capillary tubes. In all panels, the C-star mixture has been replaced with an aqueous solution of 25 mM calcein to aid visibility. (A-E) Key steps, in order, to be taken prior to annealing, corresponding to protocol sections 2.1 and 2.2. (F-J) Key steps, in order, to be taken after annealing, corresponding to protocol section 2.3. During extraction (panels (I-J)), DNA condensates will sediment from the capillary into the buffer reservoir as long as the microcentrifuge tube is stored vertically. Condensates will not be visible to the naked eye. <a href="https://www.jove.com/files/ftp_upload/66738/66738fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Preparation of C-star mixtures for single-component condensates 
	NOTE: Sequence details for various designs of single components can be found in Supplementary Table 1 and across previous works29,30,32. The slow annealing protocols defined in steps 2.1.9 and 2.2.2 were developed to ensure the relaxation of the nanostar networks into compact morphologies, given the strongly temperature-dependent rheological properties of nanostar networks37.
	<ol>
		<li>Oligonucleotide and buffer components will vary depending on the C-star design desired. Prepare a 60 &#181;L mixture of single-component C-star condensates at 5 &#181;M concentration for either the non-responsive four-arm C-star, the TMSD-responsive C-star, or the RNA-templating C-star.</li>
		<li>For each design, pipette the appropriate components listed in Supplementary Table 2 into a separate microcentrifuge tube and mix thoroughly via pipetting. 
		NOTE: All oligonucleotide stock solutions are assumed to be at 100 &#181;M concentration in TE.</li>
		<li>Withdraw the entire 60 &#181;L of C-star solution and pipette carefully to inject the mixture into the cleaned, dry glass capillary tube as shown in Figure 2A, taking care to avoid the introduction of air bubbles.</li>
		<li>Using a pipette, inject approximately 9-12 &#181;L of mineral oil into each end of the capillary tube, capping the sample such that there is no free interface between the C-star solution and the air (see Figure 2B). Then, carefully dry the capillary tube with tissue paper to ensure no oil is present on the outside of it (Figure 2C), taking care not to wick mineral oil or C-star solution out of the capillary tube. The end result is demonstrated in Figure 2D.</li>
		<li>Prepare a small batch of two-part epoxy glue to adhere each end of the capillary tube, flat side down, to a glass cover slip. Ensure the openings of the tube are completely covered in glue, forming a continuous seal, as shown in Figure 2E.</li>
		<li>Set aside to cure for a minimum of 3 h, but preferably overnight.</li>
		<li>After ~30 min of curing, inspect the glue layer for the presence of gaps in the seal caused by air bubbles. If these are present, seal them using a small quantity of glue.</li>
		<li>Wrap the capillary tube glued to the coverslip in tin foil, ensuring the foil is kept flat at the underside of the glass cover slip. The foil ensures good thermal contact between the same and the heating element of the thermal cycler (see next step).</li>
		<li>Place the wrapped sample into a thermal cycler and anneal using the following protocol: Hold at 95 &#176;C for 30 min, then cool from 85 &#176;C to 50 &#176;C at -0.04 &#176;C&#183;min-1, and then cool from 50 &#176;C to RT at -0.5 &#176;C&#183;min-1.
		<ol>
			<li>Store the annealed C-star condensates at 4 &#176;C or RT for extended periods (months) as long as the capillary remains sealed, as the thermal annealing effectively sterilizes the sample and denatures nucleases. Keep the capillary flat during storage and handling to prevent condensates from sedimenting towards one end and aggregating.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of C-star mixtures for binary condensates 
	&#8203;NOTE: To inform the selection of C-star populations for binary systems, refer to the work by Malouf et al., which describes the design rules and expected phase behavior of binary C-star condensates30.
	<ol>
		<li>To prepare binary condensates, combine 30 &#181;L volumes of the C-star mixtures described in step 2.1 above to a total volume of 60 &#181;L, mix thoroughly, load into capillaries, and follow steps 2.1.4-2.1.8.</li>
		<li>Place the wrapped sample into a thermal cycler and anneal using the following protocol: Hold at 95 &#176;C for 30 min, then cool from 85 &#176;C to 40 &#176;C at -0.01 &#176;C&#183;min-1, and then cool from 40 &#176;C to RT at -0.1 &#176;C&#183;min-1.</li>
	</ol>
	</li>
	<li>Extraction of C-star condensates from capillary tubes.
	<ol>
		<li>Prepare a large (1.5 mL or 2.0 mL) microcentrifuge tube containing 60 &#181;L of 0.3 M NaCl in TE buffer.</li>
		<li>Unwrap the capillary sample and use a diamond scribing pen to score the underside of the coverslip at each inner end of the glued area (Figure 2F). Break off the coverslip in this region and discard appropriately.</li>
		<li>Thoroughly clean the capillary tubes with ethanol and dry.</li>
		<li>Score each end of the capillary tubes with the diamond scribing pen, first with the underside (Figure 2G), then flipping and scoring the tubes &#34;right side up&#34;.
		<ol>
			<li>Ensure that the score lines do not overlap the oil-water interface. They must be within the aqueous region to prevent oil from remaining in the extracted sample.</li>
		</ol>
		</li>
		<li>Snap off the ends of the capillary tubes (Figure 2H), retaining the central part of the tube and discarding the rest. Place the cut capillary tube into the microcentrifuge tube prepared in step 2.3.1, ensuring that the bottom of the capillary tube makes contact with the buffer solution (Figure 2I).</li>
		<li>The condensates will sediment from the tube into the buffer reservoir through gravity; allow this to take place for a minimum of 10 min (Figure 2J). Remove the cut capillary tube and discard it appropriately. This gentle extraction approach limits the risk of condensate aggregation.</li>
	</ol>
	</li>
</ol>
3. Transcription of an RNA aptamer from RNA-templating C-star condensates
NOTE: For the production of the Broccoli RNA aptamer, a solution of difluoro-4-hydroxybenzylidene imidazolidinone (DFHBI) is required - DFHBI powder is first prepared as a stock solution at 10 mM in dimethyl sulfoxide (DMSO), which is then diluted to 600 &#181;M in RNase- and DNase- free water.
<ol>
	<li>Washing RNA-templating C-star condensates 
	NOTE: RNA-templating C-star condensates are washed three times to ensure unbound template oligonucleotides are removed.
	<ol>
		<li>Allow the solution of extracted condensates to settle for at least 5 min, and then remove approximately half the volume of the supernatant.
		<ol>
			<li>For a 60 &#181;L volume of extracted condensates, remove between 25-30 &#181;L of the supernatant.</li>
			<li>Pipette from the top of the liquid level to minimize the number of condensates removed during this step.</li>
		</ol>
		</li>
		<li>Add the same volume of 0.3 M NaCl in TE to replace the removed supernatant and mix by pipetting.</li>
		<li>Repeat the previous two steps for a total of three cycles.</li>
	</ol>
	</li>
	<li>Preparation of T7 transcription mixture 
	&#8203;NOTE: The T7 transcription mixture is prepared using the CellScript T7-FlashScribe Transcription Kit. Step 3.2.2&#160;is a modification of the manufacturer&#39;s protocol, which can be found here38. Here we describe the transcription of the Broccoli RNA aptamer, which induces fluorescence in DFHBI upon binding. For other light-up aptamers, replace the volume of DFHBI described in step 3.2.2 with the appropriate fluorogen. For other transcripts, remove this component entirely.
	<ol>
		<li>Prepare a stock solution of 10 mM DFHBI in DMSO, then dilute an aliquot to an end concentration of 600 &#181;M using RNase- and DNase- free water.</li>
		<li>Defrost the transcription kit components on ice and then pipette the following into an autoclaved microcentrifuge tube at room temperature using sterile pipette tips: 2 &#181;L of 10x T7-Transcription Buffer (provided in the transcription kit), 1.8 &#181;L of 100 mM ATP, 1.8 &#181;L of 100 mM CTP, 1.8 &#181;L of 100 mM UTP, 1.8 &#181;L of 100 mM GTP, 2 &#181;L of 100 mM DTT, 2 &#181;L of 600 &#181;M DFHBI, 0.5 &#181;L of RNase inhibitor, 2 &#181;L of T7-enzyme solution (provided in the transcription kit).</li>
		<li>Gently mix the solution by pipetting.</li>
		<li>Use transcription mixture for step 3.3 (synthesis of RNA transcript) immediately after preparing.</li>
	</ol>
	</li>
	<li>Synthesis of RNA transcript
	<ol>
		<li>Into a chamber suitable for microscopy imaging, pipette 3.3 &#181;L of the washed condensates prepared from RNA-templating C-stars and add the total volume of the transcription mixture prepared prior.</li>
		<li>Acquire microscopy images for a duration of 18 h, starting immediately after adding the transcription mixture to the condensates. 
		NOTE: Microscopy settings will depend on the system prepared. Ensure that the appropriate excitation and emission settings are used for any fluorophores in the sample, along with exposure times that will not lead to saturation as transcription progresses. As for any time-lapse, a compromise should be found between a high enough laser or LED power for a good signal while also minimising the risk of photobleaching. Photobleaching over long periods of time will be minimised on LED-based systems compared to laser-based systems. Suggested imaging intervals: 20 min for the first 2 h, and 30 min subsequently.</li>
	</ol>
	</li>
</ol>

Versatile Biomolecular Condensates from Amphiphilic DNA Nanostars or C-Stars

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-controlled+adhesion+and+microemulsification+in+a+two-phase+system+of+DNA+liquid+droplets.">Sequence-controlled adhesion and microemulsification in a two-phase system of DNA liquid droplets.</a> J Phys Chem. 124 (40), 8888-8895 (2020).</li><li>Sato, Y., Sakamoto, T., Takinoue, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-based+engineering+of+dynamic+functions+of+micrometer-sized+DNA+droplets.">Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets.</a> Sci Adv. 6 (23), 3471 (2020).</li><li>Saleh, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuole+dynamics+and+popping-based+motility+in+liquid+droplets+of+DNA.">Vacuole dynamics and popping-based motility in liquid droplets of DNA.</a> Nat Commun. 14 (1), 3574 (2023).</li><li>Rubio-S&#225;nchez, R., Fabrini, G., Cicuta, P., Michele, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiphilic+DNA+nanostructures+for+bottom-up+synthetic+biology.">Amphiphilic DNA nanostructures for bottom-up synthetic biology.</a> Chem Commun. 57 (95), 12725-12740 (2021).</li><li>Brady, R. A., Brooks, N. J., Cicuta, P., Di Michele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+amphiphilic+DNA+C-Stars.">Crystallization of amphiphilic DNA C-Stars.</a> Nano Lett. 17 (5), 3276-3281 (2017).</li><li>Leathers, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction-diffusion+patterning+of+DNA-based+artificial+cells.">Reaction-diffusion patterning of DNA-based artificial cells.</a> J Am Chem Soc. 144 (38), 17468-17476 (2022).</li><li>Malouf, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sculpting+DNA-based+synthetic+cells+through+phase+separation+and+phase-targeted+activity.">Sculpting DNA-based synthetic cells through phase separation and phase-targeted activity.</a> Chem. 9 (11), 3347-3364 (2023).</li><li>Fabrini, G., Minard, A., Brady, R. A., Antonio, M. D., Michele, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation-responsive+and+photocleavable+hydrogels+from+noncanonical+amphiphilic+DNA+nanostructures.">Cation-responsive and photocleavable hydrogels from noncanonical amphiphilic DNA nanostructures.</a> Nano Lett. 22 (2), 602-611 (2022).</li><li>Brady, R. A., Brooks, N. J., Foder&#224;, V., Cicuta, P., Di Michele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiphilic-DNA+platform+for+the+design+of+crystalline+frameworks+with+programmable+structure+and+functionality.">Amphiphilic-DNA platform for the design of crystalline frameworks with programmable structure and functionality.</a> J Am Chem Soc. 140 (45), 15384-15392 (2018).</li><li>Walczak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+core-shell+DNA+particles+trigger+lipid-membrane+disruption+and+bacteria+entrapment.">Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment.</a> Nat Commun. 12 (1), 4743 (2021).</li><li>Zhang, D. Y., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+DNA+strand+displacement+kinetics+using+Toehold+exchange.">Control of DNA strand displacement kinetics using Toehold exchange.</a> J Am Chem Soc. 131 (47), 17303-17314 (2009).</li><li>. Integrated DNA Technologies OligoAnalyzer Tool Available from: <a target="_blank" href="https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos">https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos</a> (2024)</li><li>Cavaluzzi, M. J., Borer, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+UV+extinction+coefficients+for+nucleoside-5'-monophosphates+and+unpaired+DNA+and+RNA.">Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA.</a> Nucleic Acids Res. 32 (1), e13 (2004).</li><li>Lattuada, E., Caprara, D., Piazza, R., Sciortino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+uniform+dynamics+in+equilibrium+colloidal+gels.">Spatially uniform dynamics in equilibrium colloidal gels.</a> Sci Adv. 7 (49), (2021).</li><li>. T7-FlashScribeTM Transcription Kit Available from: <a target="_blank" href="https://www.cellscript.com/products/018pl0617CS.pdf">https://www.cellscript.com/products/018pl0617CS.pdf</a> (2017)</li><li>Zadeh, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NUPACK:+Analysis+and+design+of+nucleic+acid+systems.">NUPACK: Analysis and design of nucleic acid systems.</a> J Comput Chem. 32 (1), 170-173 (2011).</li><li>Walczak, M., Brady, R. A., Leathers, A., Kotar, J., Di Michele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+hydrophobic+moieties+on+the+crystallization+of+amphiphilic+DNA+nanostructures.">Influence of hydrophobic moieties on the crystallization of amphiphilic DNA nanostructures.</a> The Journal of Chemical Physics. 158 (8), 084501 (2023).</li></ol>

No conflicts of interest declared.

The protocol described here provides an approach for the preparation of one- or two- component condensates from amphiphilic DNA nanostars, with design variations to introduce different responses into the condensates. The given protocol produces condensates in a buffer solution of 0.3 M NaCl in TE, but the buffer conditions can be amended by appropriately modifying the volumes listed above. Previous work has studied the formation of C-star condensates in 0.2 M NaCl in TE and 0.1 M NaCl in TE and in phosphate-buffered sali...

Synthetic cells are micrometer-scale (10-50 &#181;m) devices constructed from the bottom-up to replicate functions and structures of extant biological cells1,2. Synthetic cells are often bound by membranes constructed from lipid bilayer vesicles3,4,5,6,7, polymersomes8,9, or proteinosomes10,11, which can also be used to establish internal compartmentalisation12,13. Inspired by the membrane-less organelles known to sustain various functionalities in living cells14, structures such as polymer coacervates, biomolecular condensates, and hydrogels are gaining traction as versatile and robust alternatives to establish both external and internal compartmentalization in synthetic cells15,16,17,18.
Leveraging the versatile toolkit of DNA nanotechnology19, multiple solutions have been developed to engineer synthetic droplets and condensates from the self-assembly of artificial DNA nanostructures, whose size, shape, functionality, valency, and mutual interactions can be precisely programmed20. DNA droplets or condensates are biocompatible and can act as scaffolds for both synthetic cells and organelles, hosting chemical and biomolecular reactions21, computing information22,23, capturing and releasing cargoes24,25, and sustaining structural responses26.
Among the diverse designs of condensate-forming DNA nanostructures, amphiphilic DNA nanostars - dubbed C-stars - have proven robust and versatile27. C-stars are simple branched motifs consisting of a fixed DNA junction (typically four-way), from which double-stranded (ds)DNA arms emerge28. The arms are then tipped with hydrophobic moieties, typically cholesterol, rendering the nanostructures amphiphilic and driving their condensation following a straightforward one-pot annealing. C-star condensates afford precise structural and functional programmability, including the possibility of establishing multi-compartment architectures29,30, structurally responding to DNA and cation triggers31, synthesizing macromolecules29, capturing and releasing payloads32, and interacting with live cells33. Below, we will describe and discuss protocols to produce C-star condensates starting from their constituent oligonucleotides.
The protocol summarizes the preparation of unary (one-component) and binary (two-component) condensates, utilizing three different C-star designs (Figure 1) -&#34;Non-responsive&#34;, &#34;TMSD-responsive&#34;, and &#34;RNA-templating&#34;. The &#34;Non-responsive&#34; C-star (panel A) consists of four &#34;core strands&#34; with distinct sequences forming the four-way junction. Four identical cholesterol-modified oligonucleotides are connected to the junction, ensuring that a cholesterol molecule is present at the end of each arm. The non-responsive C-stars constitute simple, inert scaffolds for unary and binary condensates. In the &#34;TMSD-responsive&#34; C-star (panel B), the connection between the cholesterolised strands and the junction is ensured by a &#34;Toeholding bridge&#34; strand, which features a dangling single-stranded (ss)DNA &#34;toehold&#34; domain. In the presence of an invader DNA strand with a complementary toehold domain, a toehold-mediated strand displacement reaction can be triggered34, whereby the invader displaces the Toeholding bridge, breaking the connection between the junction and the hydrophobic moieties and triggering the disassembly of the DNA network32. Finally, the &#34;RNA templating&#34; C-star (panel C) includes a &#34;Base&#34; modification complementary to a &#34;Bridge&#34; strand, the latter of which links the transcribable ssDNA template for the Broccoli aptamer29. Sequence details of the constituent oligonucleotides for the three types of C-star designs mentioned here can be found in Supplementary Table 1 and across previous works29,30,32.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66738/66738fig01v2.jpg" /> 
Figure 1. Schematics of three different designs of amphiphilic DNA nanostars (C-stars). Oligonucleotide sequences for various examples of the C-stars described here can be found in Supplementary Table 1. (A) Schematic of a C-star designed to form non-responsive condensates, with the component oligonucleotide strands &#34;Core 1&#34;, &#34;Core 2&#34;, &#34;Core 3&#34;, &#34;Core 4&#34;, (coloured in shades of pink) and &#34;Terminal cholesterol&#34; (coloured in blue). Each unique colour represents an oligonucleotide strand of unique sequence. &#34;Core 1&#34; and &#34;Core 3&#34; are each partially complementary to &#34;Core 2&#34; and &#34;Core 4&#34;, but non-complementary to each other. (B) Schematic of a C-star designed to disassemble upon the addition of an invading strand via&#160;toehold-mediated strand displacement, as described in previous work32. This C-star is composed of &#34;Core&#34; and &#34;Terminal cholesterol&#34; strands (coloured in grey) as well as a &#34;Terminal complement&#34; (shown in orange) and a &#34;Toeholding bridge&#34; strand (shown in dark teal). The latter contains a six-nucleotide overhang to which an appropriately designed invader strand can bind and subsequently entirely displace the &#34;Toeholding bridge&#34; strand, which causes the dissociation of the central nanostar junction (composed of &#34;Core 1, 2, 3, and 4&#34;) from the duplexes composed of the &#34;Terminal complement&#34; and &#34;Terminal cholesterol&#34; strands. (C) Schematic of a C-star functionalised with a DNA template for an RNA aptamer. This, too, is composed of the &#34;Terminal cholesterol&#34; strand and &#34;Core 2, 3, and 4&#34; (all shown in grey), as well as an extended version of the &#34;Core 1&#34; strand (shown in pink), a &#34;Base&#34; strand (brown), a &#34;Bridge&#34; strand (yellow), and the &#34;Aptamer template&#34; (green). The DNA duplex composed of the latter two strands forms the T7 polymerase promoter region, which marks the transcription start site. <a href="https://www.jove.com/files/ftp_upload/66738/66738fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
C-star condensates form upon thermal annealing of the constituent oligonucleotides, which in the protocol presented here is conducted within sealed glass capillaries with a high aspect ratio rectangular cross-section. These containers offer multiple key advantages: i) Sealing ensures that evaporation is completely prevented over the (sometimes slow) annealing steps; ii) The optical-quality flat bottom of the capillaries enables imaging of the self-assembly (or disassembly) transient; iii) the high aspect ratio of the capillaries ensures that heavy condensates settle over a wide, flat area, reducing chances of coalescence and aggregation at later stages of the self-assembly transient that would occur in wedge-shaped containers (e.g., microcentrifuge tubes), and producing relatively monodisperse condensate populations; iv) performing the annealing in an elongated glass capillary minimizes exposure of the sample to hydrophobic interfaces (air, plastic or oil), which have been observed to perturb self-assembly by recruiting the amphiphilic cholesterolised oligonucleotides. Once the assembly protocol is completed, condensates can be extracted from glass capillaries for further experiments that involve additional reagents.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m syringe filters</td>
			<td>Sigma-Aldrich</td>
			<td>SLGVR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 60 mm #1.5 Rectangular cover glasses, Menzel Gl&#228;ser</td>
			<td>VWR</td>
			<td>631-0853</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td>34683</td>
			<td></td>
		</tr>
		<tr>
			<td>6 L Ultrasonic Cleaner with Digital Timer and Heat, 230 VAC</td>
			<td>Cole-Parmer</td>
			<td>WZ-08895-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Araldite Rapid Adhesive 2 Part Epoxy Glue</td>
			<td>RS</td>
			<td>ARA-400005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad C1000 thermal cycler</td>
			<td>Bio-Rad</td>
			<td>1851197</td>
			<td></td>
		</tr>
		<tr>
			<td>Brand Microcentrifuge Tube 2 mL with Locking Lid</td>
			<td>Fisher Scientific</td>
			<td>15338665</td>
			<td>2 mL microcentrifuge tubes for the extraction of C-star condensates</td>
		</tr>
		<tr>
			<td>Diamond Scribing Pen</td>
			<td>RS</td>
			<td>394-217</td>
			<td></td>
		</tr>
		<tr>
			<td>Difluoro-4-hydroxybenzylidene imidazolidinone (DFHBI)</td>
			<td>Sigma-Aldrich</td>
			<td>SML1627</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf PCR Clean Colorless Safe-Lock Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>0030123301</td>
			<td>0.5 mL microcentrifuge tubes for the preparation of C-star mixtures</td>
		</tr>
		<tr>
			<td>Ethanol Absolute 99.8+%</td>
			<td>Fisher Scientific</td>
			<td>10437341</td>
			<td>70% ethanol is sufficient for cleaning purposes</td>
		</tr>
		<tr>
			<td>Fisherbrand ZX4 IR Vortex Mixer</td>
			<td>Fisherbrand</td>
			<td>13284769</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III</td>
			<td>Hellma</td>
			<td>9-307-011-4-507</td>
			<td></td>
		</tr>
		<tr>
			<td>Hollow Rectangle Capillaries ID 0.40 x 4.00 mm, 50 mm in length</td>
			<td>CM Scientific</td>
			<td>2540-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>69794</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge, 230 V</td>
			<td>PRISM(TM)</td>
			<td>Z763128</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td>Used to measure absorbance of oligonucleotides for concentration calculations</td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>Oligonucleotide sequences are unique to the C-star design required.</td>
		</tr>
		<tr>
			<td>ScriptGuard RNase inhibitor</td>
			<td>CELLSCRIPT</td>
			<td>C-SRI6310K</td>
			<td>RNase inhibitor</td>
		</tr>
		<tr>
			<td>T7-FlashScribe Transcription Kit</td>
			<td>Cambio</td>
			<td>C-ASF3507</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-EDTA buffer, 100x stock solution</td>
			<td>Sigma-Aldrich</td>
			<td>574793</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977035</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Spec-Wipe 3 Wipers</td>
			<td>VWR</td>
			<td>21914-758</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthetic condensates and cell-like architectures from amphiphilic dna nanostructures

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

After annealing, C-star condensates can be imaged directly in the capillary tube, or after extraction, to confirm their formation. For all C-star design variations, one should observe distinct spherical or polyhedral condensates approximately 10-50 &#181;m in diameter, the latter forming when crystallization occurs28,32. For single-component condensates, the condensates should be discrete and uniform in appearance, and may appear polyhedral (...

Author Spotlight: Developing Synthetic Cells from Programmable Amphiphilic DNA Nanostructures

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be ...

synthetic-condensates-cell-like-architectures-from-amphiphilic-dna

Research

JoVE Journal

Bioengineering

640 Views.  University of Cambridge. We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template. 

Video: Author Spotlight: Developing Synthetic Cells from Programmable Amphiphilic DNA Nanostructures

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

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 ...

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 ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...