Engineering circular RNA for enhanced protein production
) were synthesized by cloning polymerase chain reaction products or pre-made DNA fragments into a custom entry vector
CircRNA plasmids were assembled by cloning parts 1–6 into a custom backbone via a second Golden Gate reaction. The BsaI Golden Gate reaction was set up and performed following the manufacturer’s instructions . Turbo Competent cells were transformed using 2 µl of the reaction and plated onto kanamycin agar plates. Non-green colonies were picked, mini-prepped and sequenced.CircRNA synthesis
To isolate circRNAs, column-purified RNA was digested with one unit of RNaseR per microgram of RNA for 60 minutes at 37 °C with shaking at 1,000 r.p.m. Samples were then column purified, quantified using a NanoDrop One spectrophotometer and verified for complete digestion using an Agilent TapeStation. In some instances, due to reagent shortages, verification was performed with agarose gel under formamide-based denaturing conditions .
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