Development and Evaluation of a Fluorescent Activated Droplet Sorting Regulatory Assay for Ribosomal Cis-Regulatory RNAs

Abstract

Existing methods of assaying the function of cis-regulatory RNAs come with significant drawbacks when assaying large RNA libraries. Highly sensitive cell-based assays such as the β galactosidase assay are labor intensive, difficult to scale up and may lose sensitivity with increased throughput. GFP and luciferase reporters can be used with FACS to increase assay throughput, but sorting small bacterial cells is challenging and greatly reduces assay sensitivity. Conversely, in vitro methods allow for fast screening of very large RNA libraries, but only select for properties of binding, not regulation. By combining the principles of classic in cell regulatory assays with modern tools, cis-regulatory RNAs can be quickly screened for regulatory activity at a large scale. The assay under development, Fluorescent Activated Droplet Sorting Regulatory Assay (FADSRA), uses microfluidics to encapsulate single cells expressing a fluorescent protein under the control of a cis-regulatory RNA. These cells are then cultured into microcolonies within the droplets, which are subsequently sorted according to fluorescent signal. Deep amplicon sequencing of the regulatory RNAs can then reveal which sequences can regulate and which cannot. Thus, FADSRA can help bridge the gap between in vitro RNA binding and gene regulation assays, providing a way to answer sophisticated questions about cis-regulatory RNAs requiring high-throughput assay methods. While many applications for FADSRA are possible, such as verifying regulatory activity of in vitro binders or screening synthetic regulators, one such application of FASDRA is the creation of fitness landscapes that probe sequence-function relationships of RNA cis-regulators. This dissertation first develops and optimizes the regulatory assay for ribosomal leaders in Chapter 2, following by creating a single mutant fitness landscape of the E. coli S15 leader RNA in Chapter 3. Results of this fitness landscape largely support previously published mutational studies and highlight the necessity of stable hairpin formation for regulation of the E. coli S15 leader. Chapter 4, examining the regulation of S15 protein and leader homologs, and Chapter 5, testing the adaptability of FADSRA to other cis-regulatory RNAs, examine possible further applications of the assay.