Synthesis and transfection of mRNAs
This protocol is extracted from research article:
Numerical operations in living cells by programmable RNA devices
Sci Adv, Aug 21, 2019; DOI: 10.1126/sciadv.aax0835

The synthesis and transfection of reporter mRNAs were performed as described previously (20). Before polymerase chain reaction (PCR) amplification of the templates for the in vitro transcription (IVT), double-stranded DNA fragments of the open reading frame of several fluorescent proteins (hmAG1, hmKO2, tagBFP, and hdKRed) and of the negative control 5′UTR and the 3′UTR were prepared via PCR with specific primers from plasmids and single-stranded oligo DNAs (table S3). Then, the fragments were mixed and amplified via fusion PCR with T7Fwd5UTR and Rev120A containing T7 promoter and poly-T tract, respectively (table S3). To synthesize the IVT templates for miRNA-responsive mRNAs, one, two, or three single-stranded oligo DNAs for the designed 5′UTRs (tables S1 and S2) were mixed into the fusion PCR instead of the negative control 5′UTR fragment. From the purified IVT templates, mRNAs were synthesized by T7 RNA polymerase (MEGAscript T7 kit, Ambion) and capped cotranscriptionally in the presence of an antireverse cap analog (New England BioLabs). In the transcription reaction, all cytidine and uridine triphosphates were replaced by 5-methylcytidine and pseudouridine triphosphates (TriLink BioTechnologies), respectively. The transcripts were purified and then treated with Antarctic Phosphatase (New England BioLabs) to remove the phosphates in the 5′ terminus. The synthesized mRNAs were further purified with RNeasy MinElute Cleanup Kit (Qiagen) and stored at −20°C until use. The mRNAs (up to 500 ng) and mirVana miRNA inhibitors (up to 6 pmol; Applied Biosystems) were transfected into the cultured cells on 24-well plates via lipofection with 1 μl of StemFect (Stemgent) according to the manufacturer’s instructions. In the first screening of 270 single-slot mRNAs and following live cell classification, the transfections were performed according to a reverse transfection protocol with 50,000 to 100,000 cells. The conditions of the mRNA transfection are summarized in table S4.

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