Measurement of the degradation rate of alphaviral RNA in BHK-21 cells
Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To determine the degradation rate of alphaviral RNA in BHK-21 cells, 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. The findings revealed that the half-life of alphaviral RNA in the cells was approximately 3.5 hours. Overall design: 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. RNA-seq was performed on a DNBSEQ-T7 platform. Cleaned paired-end reads were aligned to the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the genome of the alphaviral RNA replication system using STAR 2.7.8a. Genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gtf) was downloaded from NCBI and edited to add the annotation information of the alphaviral RNA replication system. Transcript abundances were estimated using featureCounts, then normalized to RPKM using in-house R scripts.