NUCLEIC ACID CATALYSTS: COMPARING THE MECHANISMS OF DNA AND RNA ENZYMES

Kinjal Desai, Nikita Ekhelikar, Chiraag Galaiya, Jennifer Kasbohm, Parth Kothari, Timothy McAvoy, Mahati Mokkarala, Sheena Rastogi, Lisa Scully, Allen Shih, John Yoo

Advisor: Dr. Adam G. Cassano
Assistant: Tim Howes

ABSTRACT

Based on previous research on DNA enzyme catalysis, it has been shown that divalent ions are effective in aiding in the cleavage of RNA substrate by the 10-23 DNA enzyme. In previous work by other laboratories, the 10-23 DNA enzyme was isolated by in vitro evolution and has been shown to cleave RNA effectively. It does this by binding to the RNA substrate via Watson-Crick base-pairing and, with cofactors, deprotonates the 2 ' -hydroxyl group on the RNA sugar. This experiment was designed to test the efficiency of monovalent ions as cofactors in comparison to divalent ions. The monovalent ions tested were lithium, ammonium, and potassium, compared against the divalent positive control of magnesium. These three monovalent ions were chosen based on several chemical properties, including ionic radius and metallic character, in order to determine if these traits had an impact on the function of the 10-23 DNA enzyme. The monovalent ions were used in higher concentrations than the divalent ion because past research demonstrated this was necessary to accommodate for the difference in valence shell structure. After the reactions ran to completion, polyacrylamide gel electrophoresis (PAGE) was used to interpret the results. After all the gels were analyzed, they showed that monovalent ions, as compared to divalent ions, are minimally effective at RNA cleavage. In light of the effectiveness of monovalent cations serving as catalytic cofactors for ribozymes, it seems that divalent cations play a more significant role stabilizing the DNA enzyme's catalytic conformation, and therefore these cations' function cannot be replaced by monovalent ions. This expands our understanding of the catalytic mechanism involved in the 10-23 DNA enzyme ' s cleavage of RNA substrate.