Tracking Native Tetrahymena Ribozyme Folding with Fluorescence

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• The folding of structured RNAs, such as the Tetrahymena group I intron ribozyme, is crucial for their function.
• Traditional methods for monitoring RNA folding rely on radioactivity-based assays, which present safety and logistical challenges.
• Existing assays involve cleavage of radiolabeled substrates and laborious gel electrophoresis for data analysis.

Purpose of the Study:

• To develop a more efficient and accessible fluorescence-based assay for monitoring RNA native state formation.
• To provide an alternative to radioactivity- and gel-based assays for studying ribozyme folding.
• To enable institutions without radioactive material capabilities to study Tetrahymena ribozyme folding.

Main Methods:

• A fluorescence-based assay was developed using a substrate labeled with 6-carboxyfluorescein (6FAM) and a black hole quencher (BHQ1).
• Cleavage of the substrate leads to the release of the quencher, resulting in a significant increase in fluorescence signal (approx. 30-fold).
• The new assay was compared side-by-side with the established radioactivity-based assay for monitoring Tetrahymena ribozyme folding.

Main Results:

• The fluorescence assay demonstrated good agreement with the radioactivity-based assay in monitoring Tetrahymena ribozyme folding.
• The assay showed increased uncertainty compared to the radioactivity-based method but offered greater ease and efficiency.
• The workflow simplification and elimination of radioactive materials make this assay more accessible.

Conclusions:

• The developed fluorescence assay provides a viable, efficient, and safer alternative for monitoring RNA native state formation.
• This method facilitates the study of Tetrahymena ribozyme folding and is adaptable for other ribozymes.
• The assay broadens the accessibility of RNA folding studies, particularly for institutions avoiding radioactive isotopes.