GoKawiilMolecular cloning
All cloning was performed using standard molecular techniques. Fragments for cloning were generated by PCR using Platinum SuperFi II Master Mix (Thermo Fisher Scientific) and appropriate oligonucleotides (IDT DNA) by plasmid digest using standard restriction enzymes (NEB) or synthesized as gene fragments (Twist Bioscience or IDT DNA). Fragment assemblies were constructed using NEBuilder HiFi DNA Assembly Mix (NEB) or Instant Sticky-end Ligase Master Mix (NEB). Assembled fragments were transformed into self-made chemically competent Escherichia coli DH5α cells. Correct clones were identified by plasmid preparation (Monarch Plasmid Miniprep Kit, NEB) and Sanger sequencing (Azenta) or rolling circle amplification directly on cells (Microsynth). Subsequently, plasmids were isolated using a Plasmid Maxiprep Kit (QIAGEN) and used for transfection. All sequences cloned in this study are listed in Supplementary Table 1.
Plasmid transfection
One day before transfection, cells were seeded at 3.0 × 104 cells per well for 96-well plates, 2.2 × 105 for 24-well plates, 7.5 × 105 for 6-well plates and 4.0 × 106 for 10-cm dishes. Cells were transfected using JetOptimus DNA transfection reagent (Polyplus transfection) with 75 ng DNA per well for 96-well plates, 300 ng DNA per well for 24-well plates, 1 µg DNA per well for 6-well plates, and 5 μg DNA for 10-cm dishes.
Cell culture and cell lines
HEK293T cells (a gift from the Institute of Developmental Genetics, Helmholtz Munich) were cultivated at 37 °C, 5% CO 2 in an H 2 O-saturated atmosphere, and maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco). The HEK293T split-Luc reporter cell line was generated by Cas9 cleavage at the AAVS1 locus and homology-directed integration of a donor construct containing LgBiT, the carboxy-terminal fragment of Fluc, separated by a P2A sequence, and the puromycin resistance gene. Three days after transfection, the cells were selected for 2 weeks with 2 µg ml−1 puromycin (Thermo Fisher Scientific). HEK293T cells stably expressing EGFR or IL-7Rα were generated by amplification of the EGFR sequence from Addgene plasmid 23935 (a gift from W. Hahn and D. Root), whereas IL-7Rα was synthesized (Twist Bioscience). Both coding sequences were cloned into the AAVS1 knock-in donor plasmid, transfected with AAVS1 targeting Cas9 and selected with 2 µg ml−1 puromycin. Dual-positive EGFR and IL-7Rα receptor cells were generated by cloning of IL-7Rα into an AAVS1 donor plasmid containing a blasticidin resistance gene, and cells were transfected and selected in 10 µg ml−1 blasticidin medium (Thermo Fisher Scientific).
Quantification of STV-mediated target RNA release into the cell culture supernatant
Supernatants from STV-releasing cells were collected and filtered through 0.45-µm polyvinylidene fluoride (PVDF) filters (Merck Millipore) after 48 h. RNA was extracted with a Monarch Total RNA Miniprep Kit (NEB), and isolated RNA was used as a template for quantitative PCR with reverse transcription (RT–qPCR) with a Luna Universal One-Step RT-qPCR Kit (NEB) and a primer/FAM-probe set (custom design, Metabion) specific for EGFP mRNA. The reaction was analysed on a QuantStudio 7 Flex device (Thermo Fisher Scientific).
Integration of diffusion-designed symmetric oligomers into STV design
Previously designed RFdiffusion symmetric oligomers were filtered for successfully assembled oligomers on the basis of size exclusion data3. In addition, all D2 symmetric oligomers were excluded. The resulting 39 sequences were synthesized (eBlocks, IDT DNA) and cloned as a C-terminal fusion to the extra STV components (PHPLC, SynL and tdPCP).
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