RNA tertiary folding monitored by fluorescence of covalently attached pyrene

The pathways by which large RNAs adopt tertiary structure are just beginnin g to be explored, and new methods that reveal RNA folding are highly desira ble. Here we report an assay for RNA tertiary folding in which the fluoresc ence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test s ystem. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide ( U107) for which derivatization at the 2'-position should not perturb the fo lded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitutio n at U107 was derivatized with a pyrene chromophore on a variable-length te ther, and then ligated to the remainder of P4-P6, providing a site-specific ally pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivati zed P4-P6 with Mg2+, the equilibrium fluorescence intensity reversibly incr eased severalfold? as expected if the probe's chemical microenvironment cha nges as the RNA to which it is attached folds. The concentration and specif icity of divalent ions required to induce the fluorescence change (Mg2+ app roximate to Ca2+ > Sr2+) correlated well with biochemical folding assays th at involve nondenaturing gel electrophoresis. Furthermore. mutations in P4- P6 remote from the chromophore that shifted the Mg2+ folding requirement on nondenaturing gels also affected in a predictable way the Mg2+ requirement fur the fluorescence increase. Initial stopped-flow studies with milliseco nd time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a singl e site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equili brium progress and the real-time kinetics of RNA tertiary folding.