Development of an automated RNA capture-SELEX for enriching modular RNA light-up sensors

Abstract

This thesis examines recent advances in selecting and functionalizing RNA aptamers for molecular sensing applications. Two related studies have been carried out: developing a robotic platform for automated RNA aptamer selection targeting small molecules and creating a modular approach to design fluorogenic RNA sensors. Together, these studies offer a framework that combines standardized aptamer discovery with adaptable sensor design. <br/> The systematic evolution of ligands by exponential enrichment (SELEX) was adapted to a robotic platform, minimizing manual intervention and enhancing reproducibility. Traditional SELEX often involves immobilizing small-molecule targets, which can alter ligand properties. To address this, capture-SELEX was used, in which RNA libraries are immobilized via hybridization to captureoligodeoxynucleotides (ODNs), and unmodified ligands in solution facilitate the recovery of bound sequences. By optimizing the robotic system, a preferential immobilization strategy for the library was systematically investigated, resulting in more substantial enrichment of binding sequences. The platform completes up to twelve selection cycles in 72 hours and has successfully enriched aptamers for several small molecules, including neomycin B, theophylline, and riboflavin. Interaction analysis using fluorescence polarization and isothermal titration calorimetry confirmed specific binding properties of enriched aptamers, with affinities in the micromolar range. Although the current capture-SELEX protocol used on the robotic system has limitations in the affinity of the enriched aptamers compared to immobilization-based selections, it offers a standardized, high-throughput method for the rapid assessment of the utility of aptamers for small molecules. <br/> In the second part of this work, the capture-SELEX strategy was extended to enable the selection of modular allosteric RNA sensors. RNA libraries were designed to couple ligand-binding aptamers to fluorogenic RNA scaffolds, enabling molecular recognition to be directly translated into fluorescence output. Using this library in capture-SELEX, aptamers that bind thiamine pyrophosphate (TPP) were identified. These aptamers were then fused to Broccoli and its red-shifted variant, Red Broccoli, to develop ligand-responsive sensors. By optimizing the linker and spacer regions, the signal-to-background ratio was improved. The selected sensors demonstrated specificity for TPP and thiamine monophosphate, with minimal response to thiamine or unrelated nucleotides. The modular design facilitated the easy swapping of fluorogenic domains and the adjustment of sensor properties, demonstrating its effectiveness for rapid and efficient development of RNA-based sensors. <br/> Overall, this thesis establishes a scalable framework for RNA aptamer discovery and deployment. By integrating automated selection with modular sensor engineering, the presented approach provides a generalizable strategy for developing RNA-based sensing systems applicable to biosensing, synthetic biology, and molecular diagnostics.