Matthew  Zaborowicz,   '11 76
   Major: Biochemistry
   Hometown: Sterling Hts., MI

Sponsor(s): Christopher Rohlman
Support: FURSCA

Abstract: 
Group I Introns are regions of ribonucleic acid enzymes also known as ribozymes that can be spliced from the surrounding RNA as the RNA acts as an enzyme on itself. In order to study the kinetics of these Group I Introns, fluorescence resonance energy transfer (FRET) is often used. FRET analysis for Group I Introns requires that a short RNA polymer have two fluorescent tags bound to on either side. Current protocols for tagging these RNA polymers appear inefficient, requiring a large ratio of the fluorescent tag (the fluorophore) relative to the RNA substrate. It is apparent the reaction has not been optimized for the wide range of RNA polymers which are commonly used in the laboratory. These fluorophores are expensive organic molecules and it would much more efficient if the amount of fluorophore could be reduced, minimizing chemical waste and the cost of materials. The purpose of this research is to optimize the reaction of RNA polymers designed to bind to the Tetrahymena and Anabaena ribozymes with the fluorophore tetramethylrhodamine and thereby reduce cost and waste incurred by this process in the lab. Beginning with the standard protocol, a number of reactions were conducted varying the reaction conditions. Reversed-phase high performance liquid chromatography (HPLC) was utilized to determine the amount of excess fluorescent tag, tetramethyl rhodamine. After testing these conditions, it was found that the reaction does indeed use a surplus of tetramethylrhodamine. We found that the amount of this reactant can be reduced to a 1:1 ratio with the RNA polymer and still provide the maximum amount of product allowing for reduced costs and less waste in future experiments.

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