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.