A FACS-based screening strategy to assess sequence-specific RNA-binding of Pumilio protein variants in E. coli

Abstract

Sequence-specific and programmable binding of proteins to RNA bears the potential to detect and manipulate target RNAs. Applications include analysis of subcellular RNA localization or post-transcriptional regulation but require sequence-specificity to be readily adjustable to any target RNA. The Pumilio homology domain binds an eight nucleotide target sequence in a predictable manner allowing for rational design of variants with new specificities. We describe a high-throughput system for screening Pumilio variants based on fluorescence-activated cell sorting of E. coli. Our approach should help optimizing variants obtained from rational design regarding folding and stability or identifying new variants with alternative binding modes.