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 necess...

Versatile Biomolecular Condensates from Amphiphilic DNA Nanostars or C-Stars

<ol>
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J., Li, M., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+construction+of+higher-order+structure+and+function+in+proteinosome-based+protocells.">Design and construction of higher-order structure and function in proteinosome-based protocells.</a> J Am Chem Soc. 136 (25), 9225-9234 (2014).</li><li>Booth, R., Qiao, Y., Li, M., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+positioning+and+chemical+coupling+in+coacervate-in-proteinosome+protocells.">Spatial positioning and chemical coupling in coacervate-in-proteinosome protocells.</a> Angew Chem Int Ed Engl. 58 (27), 9120-9124 (2019).</li><li>Hindley, J. W., 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=Light-triggered+enzymatic+reactions+in+nested+vesicle+reactors.">Light-triggered enzymatic reactions in nested vesicle reactors.</a> Nat Commun. 9 (1), 1093 (2018).</li><li>Zubaite, G., Hindley, J. W., Ces, O., Elani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+reconfiguration+of+subcompartment+architectures+in+artificial+cells.">Dynamic reconfiguration of subcompartment architectures in artificial cells.</a> ACS Nano. 16 (6), 9389-9400 (2022).</li><li>Hirose, T., 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=A+guide+to+membraneless+organelles+and+their+various+roles+in+gene+regulation.">A guide to membraneless organelles and their various roles in gene regulation.</a> Nat Rev Mol Cell Biol. 24 (4), 288-304 (2023).</li><li>Guindani, C., Silva, L. C. d., Cao, S., Ivanov, T., Landfester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+cells:+From+Simple+Bio-Inspired+Modules+to+Sophisticated+Integrated+Systems.">Synthetic cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems.</a> Angew Chem Int Ed Engl. 61 (16), e202110855 (2022).</li><li>Adamala, K. P., 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=Present+and+future+of+synthetic+cell+development.">Present and future of synthetic cell development.</a> Nat Rev Mol Cell Biol. 25 (3), 162-167 (2023).</li><li>Allen, M. E., 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=Biomimetic+behaviors+in+hydrogel+artificial+cells+through+embedded+organelles.">Biomimetic behaviors in hydrogel artificial cells through embedded organelles.</a> Proc Natl Acad Sci U S A. 120 (35), e2307772120 (2023).</li><li>Cook, A. B., Novosedlik, S., Hest, J. C. M. v. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+coacervate+materials+as+artificial+cells.">Complex coacervate materials as artificial cells.</a> Acc Mater Res. 4 (3), 287-298 (2023).</li><li>Seeman, N. C., Sleiman, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+nanotechnology.">DNA nanotechnology.</a> Nat Rev Mater. 3, 17068 (2018).</li><li>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=DNA+droplets+for+intelligent+and+dynamical+artificial+cells:+from+the+viewpoint+of+computation+and+non-equilibrium+systems.">DNA droplets for intelligent and dynamical artificial cells: from the viewpoint of computation and non-equilibrium systems.</a> Interface Focus. 13 (5), 20230021 (2023).</li><li>Liu, W., Lupfer, C., Samanta, A., Sarkar, A., Walther, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switchable+hydrophobic+pockets+in+DNA+protocells+enhance+chemical+conversion.">Switchable hydrophobic pockets in DNA protocells enhance chemical conversion.</a> J Am Chem Soc. 145 (13), 7090-7094 (2023).</li><li>Wilner, O. I., Willner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+DNA+nanostructures.">Functionalized DNA nanostructures.</a> Chem Rev. 112 (4), 2528-2556 (2012).</li><li>Gong, J., Tsumura, N., Sato, Y., 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=Computational+DNA+droplets+recognizing+miRNA+sequence+inputs+based+on+liquid-liquid+phase+separation.">Computational DNA droplets recognizing miRNA sequence inputs based on liquid-liquid phase separation.</a> Adv Funct Mater. 32, 2202322 (2022).</li><li>Jeon, B. -. J., Nguyen, D. T., Saleh, O. 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. 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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. 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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.

Author Spotlight: Developing Synthetic Cells from Programmable Amphiphilic DNA Nanostructures

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Our research focuses on using programmable amphiphilic DNA nanostructures to develop synthetic cells that mimic biological cells. We're studying how and why our nanostructures self-assemble and how to make them functionally complex for potential applications like drug delivery, materials templating, and information storage. Firstly, our protocol introduces a simple and modular platform for designing synthetic cells with both structural and functional complexity.Then, annealing DNA condensating glass capillary tubes offers a robust way of producing them. And what we really like about this method is that we can image the condensates directly under a microscope. By combining our condensates with other modular building blocks, we've shown how a simple process can be used to generate complex structures without the need for expensive equipment or time-consuming synthesis and purification steps.Our work enables other researchers in the field to construct their own synthetic cells using our amphiphilic DNA nanostructures. We hope that others will take advantage of a robust, simple protocol to design responsive structures that can help them solve their own research questions.

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 (...

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 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