Studies of RNA packaging by MS2 coat proteins in E. coli

The goal of this project is to measure how RNA sequence and structure affect the selectivity with which RNA molecules are packaged into virus-like particles by bacteriophage MS2 coat proteins expressed in Escherichia coli. To do this, we developed an in-cellulo competition assay in which plasmid-derived RNA transcripts with defined sequences compete against cellular and vector transcripts for packaging by plasmid-derived coat proteins. In each experiment, E. coli cells were transformed with a plasmid encoding the MS2 coat protein and an RNA insert of interest. After 24 hours of uninduced expression, cells were lysed and the assembled particles were purified using ultracentrifugation and multiple rounds of nuclease digestion to remove unpackaged RNA. Packaged RNA was extracted from the purified particles and submitted for high-throughput sequencing on an Illumina NovaSeq platform. Sequencing reads were aligned to three references: the plasmid insert sequence, the plasmid vector sequence, and the E. coli host genome. These alignments were used to quantify the identity and relative abundance of RNA species encapsidated in each sample. The dataset includes packaged RNA from a panel of engineered constructs with systematically varied lengths, sequences, and structural features, as well as packaged RNA from natural MS2 particles produced by infection. This dataset supports broad analyses of RNA packaging behavior and provides a platform for testing how specific sequence or structural features influence encapsidation in a cellular environment.