Sensing Levofloxacin with an RNA Aptamer as a Bioreceptor.

To combat the growing threat of antibiotic resistance, environmental testing for antibiotic contamination is gaining an increasing role. This study aims to develop an easy-to-use assay for the detection of the fluoroquinolone antibiotic levofloxacin. Levofloxacin is used in human and veterinary medicine and has been detected in wastewater and river water. An RNA aptamer against levofloxacin was selected using RNA Capture-SELEX. The 73 nt long aptamer folds into three stems with a central three-way junction. It binds levofloxacin with a Kd of 6 µM and discriminates the closely related compound ciprofloxacin. Furthermore, the selection process was analyzed using a next-generation sequencing approach to better understand the sequence evolution throughout the selection. The aptamer was used as a bioreceptor for the development of a lateral flow assay. The biosensor exploited the innate characteristic of RNA Capture-SELEX to select aptamers that displace a complementary DNA oligonucleotide upon ligand binding. The lateral flow assay achieved a limit of visual detection of 100 µM. While the sensitivity of this assay constrains its immediate use in environmental testing, the present study can serve as a template for the selection of RNA aptamer-based biosensors.

Efficient Method to Identify Synthetic Riboswitches Using RNA-Based Capture-SELEX Combined with In Vivo Screening.

Synthetic riboswitches are a promising tool for conditional gene expression. In vitro selected aptamers used as binding domains for the design of RNA-based switches have to exhibit excellent binding affinity as well as ligand binding-induced structural changes. Selection via Capture-SELEX favors the enrichment of aptamers which exhibit both characteristics. For the Capture-SELEX, an RNA pool is used that gets immobilized onto a capture oligonucleotide by hybridization. Addition of the ligand frees the aptamers by their binding to the ligand, resulting in the release from the capture oligonucleotide through structural changes. These sequences get reverse transcribed, PCR amplified, and used for the following selection rounds. In this publication, we present a detailed protocol for Capture-SELEX, followed by screening in yeast to identify aptamers suitable for the design of synthetic riboswitches.