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Nucleic Acid Catalysts: Comparing the Mechanisms of DNA and RNA Enzymes

Nucleic Acid Catalysts: Comparing the Mechanisms of DNA and RNA Enzymes. Team 1: Kinjal D., Nikita E., Chiraag G., Jen K., Parth K., Tim M., Mahati M., Sheena R., Lisa S., Allen S., John Y. Advisors: Dr. Cassano & Tim Howes. Presentation Objectives. Background information and context

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Nucleic Acid Catalysts: Comparing the Mechanisms of DNA and RNA Enzymes

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  1. Nucleic Acid Catalysts:Comparing the Mechanisms of DNA and RNA Enzymes Team 1: Kinjal D., Nikita E., Chiraag G., Jen K., Parth K., Tim M., Mahati M., Sheena R., Lisa S., Allen S., John Y. Advisors: Dr. Cassano & Tim Howes

  2. Presentation Objectives • Background information and context • Mechanism • Data • Discussion of results • Conclusion

  3. Historical Background Enzymes are biological catalysts Natural Enzymes can be made of protein or RNA DNA enzymes are not found in nature Santoro Stephen W., Joyce Gerald F. A General Purpose RNA-cleaving DNA Enzyme. Proc. Natl. Acad. Sci. USA. [Internet]. [cited 26 Jul 2008] 94: 4262-4266. Available at http://www.pnas.org

  4. In vitro evolution led to DNA enzyme 10-23 Silverman, Scott K. Deoxyribozymes: DNA catalysts for bioorganic chemistry. Organic Biomolecular Chem. 2004 Sept; 2701-2706.

  5. In context To enhance our knowledge of the groundbreaking field of DNA enzymes To explore potential treatments for RNA viruses – e.g. HIV, Avian Flu.

  6. Mechanism: DNA slicing RNA Cleaving point GCGUGGGU AGAGAGAGG RNA substrate C A A C G A CTCTCTCC CGCACCCA G G C T A G C T A DNA Enzyme

  7. Cleaving Studies show a dependence on divalent metal ions (Mg2+, Ca2+, etc.) Divalent ions may play a structural role or participate in direct catalysis

  8. Substitutes • Previous studies with high hydrostatic pressure or high concentrations of monovalent ions • Mimic effects of divalent ion • Li+, K+, NH4+

  9. Lanes With Expected Bands Enzyme RNA substrate RNA product RNA substrate marker RNA product marker DNA enzyme marker 10 mM Mg2+ No Salt Loading Buffer 1 M Salt 2 M Salt 3 M Salt 4 M Salt

  10. RNA Reactant RNA Product DNA Enzyme Loading Buffer 1M 2M 3M 4M MgCl2 No Salt KCl LiCl NH4Cl

  11. DNA Enzyme 2M 3M No Salt RNA Reactant RNA Product Loading Buffer 1M MgCl2 KCl + EDTA LiCl + EDTA 4M NH4Cl + EDTA

  12. RNA Reactant RNA Product DNA Enzyme Loading Buffer 1M 2M 3M MgCl2 No Salt KCl + EDTA 20 Hour Incubation LiCl + EDTA 20 Hour Incubation

  13. Conclusions 10-23 DNA enzyme efficiently cleaves the RNA substrate in presence of Mg2+ EDTA eliminates cleavage by chelating divalent metal ions. High concentrations of monovalent cations do not support RNA cleavage by the 10-23 DNA enzyme. 10-23 DNA Enzyme is more dependent on the presence of divalent metal ions for activity than naturally occurring RNA enzymes

  14. Acknowledgements • Project Advisors: • Dr. Cassano • Tim Howes • Faculty: • Dr. Miyamoto • Dr. Surace • Dr. Quinn • Myrna Papier

  15. Thank You To Our Sponsors John and Laura Overdeck Jewish Communal Fund NJGSS Alumnae and Parents 1984-2008 Novartis Schering-Plough Foundation The Dorr Foundation The Edward W. and Stella C. Van Houten Memorial Fund The Jennifer A. Chalsty Foundation

  16. Questions?

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