Optimization of fluorescence based Group I ribozyme studies

Abstract

Group I introns are self‐splicing RNA's. They can be made into ribozymes through modifications which creates an active site at the location of the initial splice site. This ribozyme can then act on a substrate strand of RNA with a base sequence complementary to the active site. These ribozymes are used to study the function of the intron itself. Fluorescent tags can be attached to the substrates so that we can study their kinetic properties. A machine that can be used is a genetic sequencer. The ABI 310 sequencer operates through capillary electrophoresis. The laser is very sensitive and can detect the fluorescent substrate in the nanomolar range. The ABI 310 is also fully automated and faster than typical bandshift gel assays. Multiple samples can be made and loaded into the instrument at a time. The data gives us information for quantification of reaction completion at a certain time point. The conditions of the ribozyme induced cleavage reaction can then be altered in an attempt to further understand the intron's function. We are working to adapt this system to a range of Group I model systems. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.