Editing VLP Expression and Purification (section)

=== In vitro transcription of RNAilet7 ===

<br/>DNA containing the T7 promoter, the RNAilet7, and T7 terminator generated by PCR was used as<br/>template for in vitro transcription (Primer: RNAi-Fwd and RNAi-Rev, Table S1). The DNA was<br/>purified with the QIAquick PCR Purification Kit (Qiagen). Approximately 1000 ng of DNA per&nbsp;20 μL reaction volume was transcribed for 4 h at 37 °C with the MEGAscript High Yield&nbsp;Transcription Kit (Applied Biosystems), followed by incubation with TURBO DNase for 15 min<br/>to 1 h at 37 °C. RNA was pelleted by ammonium acetate precipitation, and the pellet was washed<br/>with 80% ethanol before drying via Speedvac or lyophilization. Pellets were resuspended in<br/>nuclease-free water and further purified by illustra™ NAP-5™ columns to remove<br/>unincorporated nucleotides.





co-expressed Qβ CP and the RNAi scaffold in E. coli resulted in&nbsp;overexpression of RNAi scaffold is overexpressed and accumulation in<br/>E. coli&nbsp; package inside VLPs .