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.

single-component-condensate-formation-from-c-star-mixtures-glass

Research

JoVE Journal

Bioengineering

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.

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 ...

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

Begin by sonicating the glass capillary tubes using an alkaline optical detergent for optical components. To do so, take the required number of glass capillary tubes and place them in a tall, narrow container, such that the capillaries do not lie flat on its base. Add a 1%alkaline detergent solution in deionized water to the container, ensuring it just covers the capillary tubes.Tap to eliminate any trapped air bubbles from within the tubes. Loosely cover the container. Place the covered container upright in the bath, ensuring the covered does not contact the bath's water.Sonicate the tubes at 40 degrees Celsius for 30 minutes before turning off the bath's heating element, then thoroughly rinse the capillaries with deionized or ultrapure water at least five times, discarding the rinsed water after each cycle. Next, add isopropanol or ethanol to the glass capillaries in the container so that the liquid level is just above the capillary tubes. After ensuring the absence of trapped air bubbles in the tubes, loosely cover the container.Sonicate the capillaries as demonstrated previously for 15 to 30 minutes. After properly disposing the alcohol, dry the capillaries under nitrogen, while handling them with lint-free tissue.

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

To begin, prepare a large microcentrifuge tube containing 60 microliters of 0.3 molar sodium chloride in tris EDTA buffer. Then unwrap the previously prepared capillary sample glued to the cover slip, and use a diamond scribing pen to score the underside of the cover slip at each inner end of the glued area. Break off the cover slip in this region and discard it appropriately.Thoroughly clean the capillary tube with ethanol before drying it. Next, score each end of the capillary tube with the diamond scribing pen, first on the underside, then flipping and scoring the tube right side up. Ensure the score lines do not overlap the oil-water interface.Snap off the ends of the capillary tubes, retaining the central part and discarding the rest. Place the cut capillary tube into the previously prepared microcentrifuge tube, ensuring the bottom of the capillary tube makes contact with the buffer solution. Allow the condensates to sediment from the tube into the buffer reservoir under gravity for a minimum of 10 minutes.Finally, remove the cut capillary tube and discard it appropriately.

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

To begin, wash the RNA-Templating C-star condensates by allowing the solution of extracted condensates to settle for at least five minutes. Then while pipetting from the top of the liquid level to minimize condensate removal, extract approximately half the volume of the supernatant. Add the same volume of 0.3 molar sodium chloride in tris-EDTA to replace the removed supernatant and mix by pipetting.Repeat the cycle of supernatant removal and buffer replacement for a total of three cycles. For the T7 transcription mixture, prepare a stock solution of 10 millimolar DFHBI in dimethyl sulfoxide. Then dilute an aliquot to a final concentration of 600 micromolar using RNAse and DNAse free water.Defrost the transcription kit components on ice. Then using sterile pipette tips, pipette the displayed components into an autoclaved microcentrifuge tube at room temperature. Gently mix the solution by pipetting and use it immediately for the synthesis of RNA transcript.To do so, pipette 3.3 microliters of the washed condensates prepared from RNA-Templating C-Stars into a chamber suitable for microscopy imaging. And add the total volume of the freshly prepared transcription mixture. Acquire microscopy images for a duration of 18 hours, starting immediately after adding the transcription mixture to the condensates.For transcription of RNA Aptamers, A successful outcome was confirmed by the increase in fluorescence of the florigen over time via microscopy.