参考文献：Chen R, Wang SK, Belk JA, Amaya L, Li Z, Cardenas A, Abe BT, Chen CK, Wender PA, Chang HY. Engineering circular RNA for enhanced protein production. Nat Biotechnol. 2023 Feb;41(2):262-272.
Cell culture and transfection：
RNA delivery was achieved with TransIT-mRNA transfection, Lipofectamine transfection or NEON electroporation. Within each experiment, the molar amount of mRNA or circRNA delivered and transfection method used was the same for all samples unless otherwise indicated. For TransIT-mRNA transfections, 3 μl of TransIT-mRNA reagent (Mirus Bio) was used per microgram of RNA. Besides this change, RNA delivery was performed following the manufacturer’s instructions.
参考文献：Wesselhoeft RA, Kowalski PS, Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat Commun. 2018 Jul 6;9(1):2629.
For all circRNA data sets presented in Fig. 2 except Cas9, 40–100 ng of RNase R-treated splicing reactions or HPLC-purified circRNAs were reverse transfected into 10,000 HEK293 cells/100 μL per well of a 96-well plate using Lipofectamine MessengerMax (Invitrogen) according to the manufacturer’s instructions.
For Cas9, 100 ng of in vitro transcribed sgRNA was reverse transfected alone or cotransfected with 150 ng of RNase R-treated Cas9 splicing reaction into 50,000 HEK293-GFP cells/500uL per well of a 24-well plate using MessengerMax.
For all RNA datasets presented in Fig. 3, equimolar quantities of each RNA (equivalent to between 40 and 80 ng dependent on size) were reverse transfected into 10,000 HEK293, HeLa, or A549 cells/100 uL per well of a 96-well plate using MessengerMax.
For experiments wherein protein expression was assessed at multiple time points, media was fully removed and replaced at each time point. Min6 cells were transfected in 96-well plate format between 60–80% confluency. Sample sizes were chosen based on pilot experiments to determine assay variance and to minimize reagent consumption while allowing for meaningful differences between conditions to be distinguished.
参考文献：Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell. 2022 May 12;185(10):1728-1744.e16.
CircRNA transfection in vitro
For circRNA transfection into HEK293T or NIH3T3 cells, 38105 cells per well were seeded in 12-well plates. Two micrograms of circRNA was transfected into HEK293T or NIH3T3 cells using Lipofectamine MessengerMax (Invitrogen) according to the manufacturer’s instructions. At 24-48 hr after transfection, the cell lysis and supernatant were collected for subsequent
detection.
参考文献：Pervasive translation of circular RNAs driven by short IRES-like elements
Cell cultures and Transfection.
293T human embryonic kidney cell line, SHSY5Y neuroblastoma cell line, and HeLa epithelial cell line were cultured in DMEM (high glucose) medium containing 10% fetal bovine serum (FBS, Hyclone).
To transient transfect plasmids into cells, 2 μg of mini-gene reporters were transfected into cells in 6 well plate, using lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction. After 48 h, cells were collected for further analysis of RNA and protein levels.
To transfect circRNAs into cells, 200 ng of RNAs were transfected into cells in 24-well plate, using lipofectamine 3000 (Invitrogen) according to the manufacturer’s instruction.
参考文献：Sensing Self and Foreign Circular RNAs by Intron Identity
Cell Culture and Transient Transfection:
1 ug of low molecular weight poly(I:C) (Invivogen, tlrl-picw), high molecular weight poly(I:C) (Invivogen, vac-pic), or interferon stimulatory DNA (Invivogen, tlrl-isdn) were transfected using Lipofectamine 2000 (Invitrogen, 11668-019). Briefly, cells were transfected at 70 to 80% confluence using Lipofectamine 2000. The nucleic acids and Lipofectamine 2000 were diluted, mixed in Opti-MEM (Invitrogen, 31985-088), and incubated for 5 min at room temperature (RT). Following, the nucleic acids and Lipofectamine 2000 were mixed together, incubated for 15 min at RT and then the nucleic acids-Lipofectamine 2000 complexes were applied to the monolayer cultures. 500 ng of linear or circRNA was transfected into one well of a 24-well plate.
参考文献：RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo
For 293 and A549 cells, 40ng of RNA was reverse transfected into 10,000 cells/100uL per well of a 96-well plate using Lipofectamine MessengerMax (Invitrogen) according to the manufacturer’s instructions, unless otherwise noted.
For HEK-Blue cells, 100ng of RNA was reverse transfected into 40,000 cells/100uL per well of a 96-well plate using Lipofectamine MessengerMax. For A549 cells transfected prior to RNA harvest and qPCR, 200ng of RNA was reverse transfected into 100,000 cells per well of a 24-well plate using Lipofectamine MessengerMax, unless otherwise noted.
For experiments wherein protein expression was assessed at multiple time points, media was fully removed and replaced at each time point. For experiments wherein SEAP activity or cytokines were analyzed, media was not replaced between transfection and assessment.
For all transfection experiments, RNA was heated to 70_C for 3 min and immediately placed on ice for 2 min prior to complexation with transfection reagent. Cell viability 36-72 h after transfection was assessed using a MultiTox kit (Promega). To detect SEAP secretion by TLR reporter and null cells, media was harvested 36-48 h after transfection and combined with HEK-Blue Detection reagent (Invivogen) to a final concentration of 1X. Media and detection reagent were incubated overnight at 37_C and then absorbance at 640nm was measured on an Infinite 200Pro Microplate Reader (Tecan). R848, polyI:C, and 3p-hpRNA were obtained from Invivogen. For TLR data in Figures 4 and 6, absorbance measured in TLR reporter cells was normalized to absorbance measured in null reporter cells containing only the plasmid with SEAP under the control of the IFN-b minimal promoter fused to five NF-kB and AP-1 binding sites for mTLR3 and hTLR8, or null reporter cells containing only the plasmid with SEAP under the control of the IL-12p40 minimal promoter fused to five NF-kB and AP-1 binding sites for mTLR7 (Invivogen).
参考文献：RNA Circularization Diminishes Immunogenicity and Can Extend Translation Duration In Vivo
参考文献：RNA circles with minimized immunogenicity as
potent PKR inhibitors
Cell culture, transfection and stimulation Human cell lines including A549, HeLa, HEK293 and 293FT cells were maintained using standard protocols from ATCC. RNAs synthesized in vitro and poly(I:C) were transfected using Lipofectamine MessengerMax reagent (Thermo) for A549, HeLa, HEK293 and 293FT cells according to the manufacturer’s protocols. About 70%–80% transfection efficiency was achieved in cells. After transfection for 1 or 6 hours, protein or total RNAs were collected for analyses.
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