Engineering a steady state FRET assay for the Twort group I ribozyme

CHED 829

Christopher S. Henes, csh10@albion.edu and Christopher E. Rohlman, crohlman@albion.edu. Department of Chemistry, Albion College, 611 E. Porter Street, Albion, MI 49224
Group I introns or ribozymes are catalytic RNAs that are capable of performing a variety of transesterification reactions including self-splicing and RNA cleavage. Their ability to self-splice is due to the presence of a guanosine substrate and a dynamic 3-dimensional conformation which forms a catalytic active site. Much of our understanding of group I introns is due to studies on the Tetrahymena group I intron. Golden, more recently has completed research with group I introns which led to development of a structural model of the group I ribozyme from the Twort virus through x-ray crystallography. Previous reports have described applications fluorescence resonance energy transfer (FRET) based and simple fluorescent probe ribozyme assays. We are currently working to develop a FRET based assay that will allow us to study Twort's folding along with its catalytic mechanism. We use both a donor and acceptor on the substrate docked in trans to the Group I ribozyme. To monitor the activity of the ribozyme we have utilized two approaches. We have developed mobility shift assays that allow us to measure ribozyme-substrate ligand binding along with ribozyme activity. To measure the kinetics of this system we have developed FRET based steady state spectoscopic assays.