Massively Parallel In Vitro Functional Analysis of Evolution-Derived Transcriptional Riboswitch Sequences

Riboswitches are structured non-coding RNA elements that regulate gene expression in response to small molecules; they serve as valuable systems in both public health and biophysical research by elucidating principles around RNA-ligand interactions, structure, and cellular function. Traditional approaches to studying riboswitches have relied on low-throughput techniques such as reporter assays or gel electrophoresis analysis of transcriptional products, which are limited in scalability. In this study, we present a high-throughput protocol to characterize the transcriptional activity of nearly 2,000 natural variants of the fluoride riboswitch in in vitro transcription. Starting with bioinformatics, we compiled a comprehensive dataset of riboswitch variants and then employed massive parallel oligonucleotide synthesis to generate an oligo pool of the riboswitch library. This pool was transcribed in vitro, converted into an Illumina-compatible next-generation sequencing (NGS) library, and analyzed to identify transcriptionally active riboswitch candidates. The workflow integrates natural riboswitch bioinformatic acquisition into a quantitative readout in a single streamlined pipeline, enabling large-scale exploration of transcriptional riboswitch function. This protocol offers a scalable method for mapping genotype-to-function relationships across transcriptional riboswitch families, accelerating the identification of functional variants for desired applications. Key features • Bioinformatics to acquire full sequences (aptamer + downstream expression platform) of naturally occurring riboswitches. • E. coli RNA polymerase in vitro transcription to characterize nearly 2,000 fluoride riboswitches and analysis with NGS.