A Modular Strategy for Development of RNA-Based Fluorescent Sensors

Abstract

Fluorescent biosensors that directly transduce binding events of small molecules into optical signals are valuable tools in the areas of therapeutics and diagnostics. However, construction of fluorescent biosensors from macromolecular receptors with desired characteristics, such as detection wavelengths and concentration ranges for ligand detection, is not a straightforward task. A ribonucleopeptide (RNP) receptor was easy to convert to a fluorescent RNP sensor without chemically modifying the nucleotide in the ligand-binding RNA. The strategy of converting the ligand-binding RNP receptor to a fluorescent RNP sensor was applied to generate fluorescent ligand- binding RNP libraries by utilizing a pool of RNA subunits obtained from the in vitro selection of ATP-binding RNPs and various fluorophore-modified peptide subunits. Simple screening of the fluorescent RNP library based on the fluorescence emission intensity changes in the absence and presence of the ligand afforded a wide variety of fluorescent RNP sensors with emission wavelengths ranged from 390 to 670 nm. Screening of the fluorescence emission intensity changes in the presence of increasing concentrations of ligand provided RNP sensors responding at wide concentration ranges of ligand. The combinatorial strategy using the modular RNP receptor enables tailoring of a fluorescent sensor for a specific ligand without knowledge of detailed structural information for the macromolecular receptor