single-component condensate formation from c-star mixtures in glass capillary tubes

Versatile Biomolecular Condensates from Amphiphilic DNA Nanostars or C-Stars

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.

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.

Single-Component Condensate Formation from C-Star Mixtures in Glass Capillary Tubes

Begin by preparing the C-Star mixtures for single component condensates using the pre-cleaned glass capillary tubes. To do so using a pipette, withdraw the entire 60 microliters of C-Star solution and transfer it carefully into the cleaned, dry glass capillary tube avoiding the introduction of air bubbles. Then, pipette approximately nine to 12 microliters of mineral oil into each end of the capillary tube to prevent a free interface between the C-Star solution and the air.Dry the outside of the capillary tube with tissue paper, ensuring no oil remains without wicking mineral oil or C-Star solution out of the tube. Use a small batch of two part epoxy glue to completely seal and adhere each end of the capillary tube, flat side down to a glass cover slip. Set aside the setup to cure for a minimum of three hours, but preferably overnight.After about 30 minutes of curing, inspect the glue layer for gaps in the seal caused by air bubbles. Wrap the capillary tube glued to the cover slip in tin foil, ensuring the foil is kept flat on the underside of the glass cover slip. Place the wrapped sample into a thermal cycler and a kneel using the specified protocol.The single component condensates were discrete, uniform and appeared polyhedral or spherical. The presence of air bubbles in the capillary tube interfered with the self-assembly of the condensates, often resulting in aggregation near the air interface.

Research

JoVE Journal

Bioengineering

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

Cleaning of Glass Capillaries for the Preparation of C-Star Condensates

Extraction of C-Star Single-Component Condensates from Glass Capillary Tubes

Transcription of an RNA Aptamer from RNA-Templating C-Star Condensates