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 RNA substrate DNA Enzyme CCGTGGAAT A CGCACCCA GCGUGGGUAGAGAGAGG CTCTCTCCCACGA Cleaving point

7. Cleaving Studies show a dependence on divalent metal ions (Mg 2+, Ca 2+, 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 +, NH 4 +

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

10. DNA Enzyme RNA Product RNA Reactant No SaltMgCl 2 2M1M3M4M LiCl NH 4 Cl Loading Buffer KCl

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

12. DNA Enzyme RNA Product RNA Reactant No SaltMgCl 2 2M1M3M LiCl + EDTA 20 Hour Incubation KCl + EDTA 20 Hour Incubation Loading Buffer

13. Conclusions 10-23 DNA enzyme efficiently cleaves the RNA substrate in presence of Mg 2+ 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