RNA is involved in vital cellular processes including transcription, protein synthesis, and catalysis. Like proteins RNA only functions once folded into its native conformation. RNA folding is complex as RNA may get kinetically trapped in inactive conformations. Therefore, catalytically-active RNA are used when studying RNA folding pathways as the onset of their activity provides a unique signal that RNA has folded to its native conformation. In our study, we used a ribozyme derived from a Group IIB intron that allows multiple turnovers of substrate. The substrate used was 21 RNA nucleotides long which contained a unique fluorescent dye on each end. The fluorescence resonance energy transfer (FRET) between dyes serves as a read out for when the ribozyme is in its native conformation as cleavage of substrate results in loss of FRET signal. Early experiments performed with excess ribozyme compared to substrate indicated the ribozyme is catalytically active as loss of FRET was observed after the pre-folded ribozyme was mixed with the substrate. Controls without ribozyme showed no similar loss of FRET signal. Future plans include changing conditions to a slight excess of substrate over ribozyme to allow multiple turnovers of cleavage to occur. The folding conditions will change to allow multiple turnovers that can be used to track productive folding of the ribozyme under near physiological conditions.

FRET-detected catalysis as a method to study RNA folding

255th National Meeting (2018)

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