Fluorescent molecular beacons mimicking RNA secondary structures to study RNA chaperone activity

Abstract

Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases. ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.