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 other link DNA Condensating G Glass capillary tubes offers a robust way of producing them, and what we really like about this method is that we can image the condensate 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 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. 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 cover 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. 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 wrap sample into a thermal cycler and a kneel using the specified protocol. The single component condensates were discreet, 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. To begin, prepare a large micro centrifuge tube containing 60 microliters of 0.3 molar sodium chloride in Tris-EDTA buffer. Then unwrap the previously prepared capillary sample, glue 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 micro centrifuge 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. 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 RNAs and DNAs free water. Defrost the transcription kit components on ice. Then using sterile pipette tips, pipette the displayed components into an autoclave micro centrifuge 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 fluengin over time via microscopy.