Improvements in Fluorescent Aptamer Biosensor Selection and their Use in Small Molecule Detection

Abstract

Fluorescent RNA-based biosensors have utility for imaging applications in living cells. These biosensors typically consist of a chromophore-binding region and a target-binding region, whereby the chromophore-binding region is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding. However, known riboswitches only exist for a limited number of molecules, significantly constraining biosensor design. To overcome this challenge, we designed a framework for producing de novo fluorescent RNA biosensors using aptamers selected from a large random library by capture-SELEX. As a proof-of-concept, we generated and characterized an RNA biosensor against L-dopa, the precursor of several neurotransmitters. We further demonstrate that aptamers selected in this fashion can be multiplexed by selecting an aptamer for DHPG, a metabolite of L-dopa, and simultaneously tracking orthogonal fluorescence output of both aptamers. We then leverage these aptamers to identify small molecule modulators of the amino acid transporter LAT1 in a fluorescence-based screen. Overall, we suggest that this approach will have utility for real-time fluorescence detection of custom targets in mammalian cells and is a powerful tool for screening effectors of small molecule transporters.