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.
Highlights
Molecular beacons (MBs) are oligonucleotide probes commonly used to target DNA for real-time monitoring of polymerase chain reactions (RT-PCRs)
Since the 50 and 30 ends are labelled with a fluorophore and a quencher, respectively, the MB acts as a switch
RNA conformational changes have been determined by the use of MBs that target specific RNA regions that become free for hybridization
Summary
Molecular beacons (MBs) are oligonucleotide probes commonly used to target DNA for real-time monitoring of polymerase chain reactions (RT-PCRs). RNA chaperone CspA of Staphylococcus aureus unfolds the RNA hairpin present in the 50UTR of its own mRNA [9] (Fig. 2a). This strategy allowed us to demonstrate that CspA unfolded the regulatory hairpin located at the cspA 50UTR and, interfered with cspA mRNA processing by RNase III. With slight modifications, this protocol may be adapted to test (1) any DNA or RNA folding structure that allows close proximity of BHQ_1 to FAM and that provides enough separation between them when disrupted; (2) the activity of RBPs such as RNA chaperones, RNA helicases and ribonucleases that target and/or process hairpin-like structures; and (3) the function of small regulatory RNAs that produce conformational changes on hairpin-like structures of their mRNA targets. E. coli BL21 (DE3) harboring pGEX-6P-2::cspA (see Note 1)
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