RNA Tertiary Folding Monitored by Fluorescence of Covalently Attached Pyrene

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Abstract
The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4−P6 domain was used as a test system. 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 folded conformation. A 15-mer RNA oligonucleotide with a 2‘-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4−P6, providing a site-specifically pyrene-labeled P4−P6 derivative. Upon titration of the pyrene-derivatized P4−P6 with Mg2+, the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg2+ ≈ Ca2+ > Sr2+) correlated well with biochemical folding assays that 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 for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4−P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.

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