Transcript Team Presentation

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
 Mechanism
 Data
 Discussion of results
 Conclusion
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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
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.
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.

Mechanism: DNA slicing RNA
Cleaving point
GCGUGGGU AGAGAGAGG
CGCACCCAGGCTAGCTACAAC GACTCTCTCC
RNA
substrate
DNA
Enzyme
Cleaving
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Studies show a dependence on divalent metal
ions (Mg2+, Ca2+, etc.)
Divalent ions may play a structural role or
participate in direct catalysis
Substitutes

Previous studies with high hydrostatic
pressure or high concentrations of
monovalent ions
Mimic
effects of divalent ion
Li+, K+, NH4+
Lanes With Expected Bands
Enzyme
RNA substrate
RNA product
Loading 1 M
Buffer Salt
2M
Salt
3M
Salt
4M
Salt
10
mM
Mg2+
No
Salt
RNA
RNA
DNA
substrate product enzyme
marker marker marker
Loading
Buffer
KCl
LiCl
NH4Cl
1M
2M
3M
4M
MgCl2 No Salt
RNA
RNA
DNA
Reactant Product Enzyme
Loading
Buffer
1M
2M
3M
MgCl2
KCl + EDTA
LiCl + EDTA
NH4Cl + EDTA
4M
No Salt
RNA
RNA
Reactant Product
DNA
Enzyme
Loading
Buffer
1M
2M
3M
MgCl2
No Salt
KCl + EDTA 20 Hour Incubation
LiCl + EDTA 20 Hour Incubation
RNA
Reactant
RNA
Product
DNA
Enzyme
Conclusions
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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
Acknowledgements

Project Advisors:
 Dr.
Cassano
 Tim Howes

Faculty:
 Dr.
Miyamoto
 Dr. Surace
 Dr. Quinn
 Myrna Papier
Thank You To Our Sponsors

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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