posted on 2023-11-01, 17:34authored byJeffrey P. Potratz, Rick Russell
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 fluorescence signal by an average
of 30-fold. A side-by-side comparison with the radioactivity-based
assay shows good agreement in monitoring Tetrahymena ribozyme folding from a misfolded conformation to the native state,
albeit with increased uncertainty. The lower precision of the fluorescence
assay is compensated for by the relative ease and efficiency of the
workflow. In addition, this assay will allow institutions that do
not use radioactive materials to monitor native folding of the Tetrahymena ribozyme, and the same strategy should be amenable
to native folding of other ribozymes.