Abstract
Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. Here we report developing a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identify new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provide conserved sequence and structure motifs that may be used for rationally guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems.
Highlights
Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types
fluorescence activated cell sorting (FACS)-Seq in mammalian cells has been used to assay the gene-regulatory effect of translational initiation start site (TIS)[31] and internal ribosomal entry site (IRES) sequence variants[32], but the process involves handling large quantities of lentiviral and cell culture volumes, which proves cumbersome for rapid iterations through the engineering design-build-test cycles
Gel-free, and massively parallel RNA-Seq approach to simultaneously measure mRNA levels associated with tens of thousands of ribozyme switch library sequences in mammalian cells
Summary
Synthetic RNA-based genetic devices dynamically control a wide range of gene-regulatory processes across diverse cell types. We report developing a quantitative, rapid and highthroughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. FACS-Seq in mammalian cells has been used to assay the gene-regulatory effect of translational initiation start site (TIS)[31] and internal ribosomal entry site (IRES) sequence variants[32], but the process involves handling large quantities of lentiviral and cell culture volumes, which proves cumbersome for rapid iterations through the engineering design-build-test cycles. The ribozyme libraries were transcribed by a U6 promoter, which could give rise to differences in transcription, RNA stability, and degradation from RNA Pol II-based mRNA expression; as such the results may not be applicable for assaying the activity of ribozyme switches for gene expression control. High-throughput and scalable strategies are critically needed to facilitate the engineering of ligand-responsive switches in mammalian cells
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