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Nucleic Acid Catalysts : The Effect of Monovalent Ions on DNA Enzymes

This study explores the impact of monovalent ions on DNA enzymes, specifically their role as cofactors in DNAzyme catalyzed reactions. The results suggest that monovalent ions are not effective cofactors in RNA cleavage, supporting the hypothesis that the function of metal ion cofactors is likely chemical rather than structural. Further research is needed to optimize gel conditions and control for other factors such as magnesium and RNA substrate.

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Nucleic Acid Catalysts : The Effect of Monovalent Ions on DNA Enzymes

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  1. Nucleic Acid Catalysts: The Effect ofMonovalent Ionson DNA Enzymes Farrah Alkhaleel, Neil Chen, Kevin Duh, Nabgha Farhat, Jonathan Huang, David Kersen, Michael McLoughlin, Connor Moseley, Adetayo Sanusi, Charissa Shen, Stefano Solari, and Brendan Wu Advisors: Dr. Adam G. Cassano, Jeremy Tang

  2. Enzymes 1878: Biological protein catalysts labeled "enzymes" Lower the Energy of activation, faster the reaction 1982: Thomas Cech discovers catalytic properties of RNA 1994: Ronald Breaker discovers  Deoxyribozymes

  3. About DNAzymes • DNAzymes do not exist in nature • Mg2+ acts as a cofactor in DNAzyme catalyzed reactions rna.berkeley.edu/Research/hdv-rbz.html

  4. Applications DNAzymes can… • Cleave RNA viruses • Be used for cardiovascular disease treatment http://www.sciencemuseum.org.uk/on-line/lifecycle/120.asp http://www.fotosearch.com/illustration/internal-heart.html

  5. Applications (continued) DNAzymes can… • prevent translation of cancerous mutations • be used to create lead detecting devices https://sci10bestq3bm.wikispaces.com/lead-gold+class+%2709 www.pdb.org/File:PBB_Protein_KRAS_image.jpg http://www.battelle.org/environment/publications/envupdates/summer2004/article7.stm

  6. DNAzyme 10-23 Created in vitro by Stephen W. Santoro and Gerald F. Joyce   www.nac.sdu.dk/Reserch/BMB/Modified_DNAzymes.htm

  7. Cleavage AGAGAGAGG GCGUGGGU l l l l l l l l l l l l l l l l CGCACCCA CTCTCTCC G G C T A G C T A C A A C G A 10-23 is highly selective, cleaving only the purine-pyrimidine bond

  8. Mechanism: Structural vs. Chemical Structural • Tertiary interaction • Charge density Ex. • Ribozymes HH, HP, and VS with monovalent ions • HDV ineffective http://commons.wikimedia.org/wiki/File:Hammerhead_ribozyme_ribbons.png http://www.chem.ucsb.edu/~molvisual/rna_biochem.html http://rna.berkeley.edu/Research/hdv-rbz.html http://www.rsc.org/chemistryworld/_denial.asp

  9. Mechanism: Structural vs. Chemical Chemical • Santoro-Joyce mechanism models Pubs.acs.org.ezproxy.drew.edu/doi/pdf/10.1021/bi9812221?cookieSet=1

  10. Divalent Ions vs. Monovalent Ions The effectiveness is already recognized Still no consensus whether structural or chemical Divalents Monovalents • Could prove that reaction only requires a concentration of positive charge http://www.hull.ac.uk/chemistry/intro_inorganic/Chap8-1NaCl.htm

  11. HYPOTHESIS If are effective in operating as a cofactor for DNAzyme 10-23, monovalent cations then the function of metal ion cofactors is rather than in nature. structural chemical

  12. Materials and Methods http://www.hull.ac.uk/chemistry/intro_inorganic/Chap8-1NaCl.htm http://www.3dchem.com/molecules.asp?ID=89 http://202.114.88.54/g/web18/wangluo/webelements.html www.dnamnd.med.usyd.edu.au/ http://www.biochemistry.ucla.edu/biochem/Faculty/Feigon/gallery.html http://www.hull.ac.uk/chemistry/intro_inorganic/Chap5-2NaCl.htm

  13. Materials and Methods

  14. Expected Results No Salt RNA Subs RNA Prod DNA Mg2+ Li+ Na+ NH4+ DNAzyme 10-23 RNA Substrate RNA Product

  15. 1st Trial and Results

  16. 2nd Trial and Results

  17. 3rd Trial and Results

  18. 3rd Trial and Results

  19. Conclusion • Monovalent ions not effective cofactors in RNA cleavage • Role of divalent ions most likely chemical

  20. Further Research • Optimal gel conditions • Control for magnesium and RNA substrate • Cobalt (III) Hexamine – Charge Density

  21. Acknowledgements Advisors • Dr. Adam G. Cassano • Jeremy Tang Faculty • Dr. Miyamoto • Dr. Surace • Dr. Quinn • Myrna Papier

  22. Acknowledgements Thank you to all our sponsors! • John and Laura Overdeck • Novartis • Jewish Communal Fund • The Ena Zucchi Charitable Trust • Fannie Mae Foundation • Bristol-Myers Squibb • Village Veterinary Hospital • Vanguard Charitable Endowment Program • NJGSS Alumnae and Parents 1984-2009

  23. Questions?

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