Tracking Native <i>Tetrahymena</i> Ribozyme Folding with Fluorescence.

ABSTRACT: Folding of the Tetrahymena group I intron ribozyme and other structured RNAs has been measured using a catalytic activity assay to monitor the native state formation by cleavage of a radiolabeled oligonucleotide substrate. While highly effective, the assay has inherent limitations present in any radioactivity- and gel-based assay. Administrative and safety considerations arise from the radioisotope, and data collection is laborious due to the use of polyacrylamide gels. Here we describe a fluorescence-based, solution assay that allows for more efficient data acquisition. The substrate is labeled with 6-carboxyfluorescein (6FAM) fluorophore and black hole quencher (BHQ1) at the 5' and 3' ends, respectively. Substrate cleavage results in release of the quencher, increasing the fluores

SUBMITTER: Potratz JP 

PROVIDER: S-EPMC10666665 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature