Direct fluoride monitoring using a fluorogenic RNA-based biosensor.

Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.

Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy.

Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.

FluorMango, an RNA-Based Fluorogenic Biosensor for the Direct and Specific Detection of Fluoride.

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen. The two modules are connected by an optimized communication module identified by ultrahigh-throughput screening, which results in extremely high fluorescence of FluorMango in the presence of fluoride, and background fluorescence in its absence. The value and efficiency of this biosensor by direct monitoring of defluorinase activity in living bacterial cells is illustrated, and the use of this new tool in future screening campaigns aiming at discovering new defluorinase activities is discussed.

High-Throughput Development and Optimization of RNA-Based Fluorogenic Biosensors of Small Molecules Using Droplet-Based Microfluidics.

Small-molecule sensing is a major issue as they can serve both in fundamental science and as makers of various diseases, contaminations, or even environment pollution. RNA aptamers are single-stranded nucleic acids that can adopt different conformations and specifically recognize a wide range of ligands, making them good candidates to develop biosensors of small molecules. Recently, light-up RNA aptamers have been introduced and used as starting building blocks of RNA-based fluorogenic biosensors. They are typically made of three domains: a reporter domain (a light-up aptamer), connected to a sensor domain (another aptamer) via a communication module. The latter is instrumental as being in charge of information transmission between the sensor and the reporting domains. Here we present an ultrahigh-throughput screening procedure to develop RNA-based fluorogenic biosensors by selecting optimized communication modules through an exhaustive functional exploration of every possible sequence permutation using droplet-based microfluidics and next-generation sequencing.