1. Nucleic Acid Catalysts: The Effect of Monovalent Ions on 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
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Enzymes are biological catalysts, essential to complex life.   They work by lowering the Gibbs' free energy necessary for the reaction to proceed through a structure known as the enzyme-substrate complex.
Discovered and defined in 1878, enzymes were originally thought to be exclusively protein structures.  This turned out to be incorrect when RNA, an amino acid, was found to have catalytic properties also.  Dr. Thomas Cech discovered and labeled RNA catalysts, calling them ribozymes.
The question then arose as to whether or not DNA, a strucurally similar compound, could also be coaxed into functioning as an enzyme.  In 1994, Professor Ronald Breaker of Yale University discovered the first known deoxyribozyme.
1994: Ronald Breaker discovers 
Deoxyribozymes

3. About DNAzymes DNAzymes do not exist in nature
Mg2+ acts as a cofactor in DNAzyme catalyzed reactions
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DNAzymes are created in vitro. Bold Mg
They require a cofactor to help in the catalytic process
The cofactor, Mg2+ being the most effective, helps to stabalize the transition state of the enzyme and forces the forward reaction.
rna.berkeley.edu/Research/hdv-rbz.html

4. Applications DNAzymes can… Cleave RNA viruses
Be used for cardiovascular disease treatment
By understanding how DNAzymes work, we can also begin to discover new applications with which we can use them.
One way that DNAzyme is being developed is for medical purposes. DNAzymes have selective cleaving capabilities allowing them to cut the nucleic acids of viruses. Researchers can design DNAzymes that are specific to the viral RNA and hope to use this technology to target single stranded RNA viruses such as the HIV virus.
DNAzymes can also be used to combat cardiovascular disease. The DNAzyme is one such deoxyribozyme and also happens to be the catalyst that we worked with. Studies using the DNAzyme have already been performed in rats where the TNF alpha mRNA was cut. This is the nucleic acid responsible for coding for the TNF alpha complex which leads to heart failure after infarction. The DNAzyme cleaved the mRNA and resulted in an almost full improvement in the rats after treatment.

5. Applications (continued)
DNAzymes can…
prevent translation of cancerous mutations
be used to create lead detecting devices
Combine the top one 
DNAzyme also has the potential to aid in cancer treatment by disrupting the translation of the mutated K-Ras(G12V) mRNA sequence. Mutated K-Ras(G12V) is responsible for the proliferation and metastasis of many types of cancers. DNAzyme can help in cancer treatment by cleaving the mRNA K-Ras(G12V) and therfore reducing the number of K-Ras(G12V) synthesized.
Another practical application for DNAzyme is in the creation of miniature  lead detecting devices that could be used for such purposes as the detection of lead in the drinking water supply.  Lead has been shown to succesfully activate RNA enzyme cleavage by DNAzyme, a property that can be utilized to detect lead.  Due to its potentially smaller size as compared to other lead detectors currently available, DNAzyme lead detectors could provide a fast, efficient, and low cost method with less waste product produced for lead detection. 

6. Created in vitro by Stephen W. Santoro and Gerald F. Joyce
DNAzyme 10-23
It was discovered in 1994 that DNA can also function as an enzyme!  
For our experiment, the enzyme we used is labeled DNAzyme   
The numbers refer to the sample set from which the DNAzyme was retrieved: "The 23rd clone after the 10th round of selective amplification."  This means,
essentially that the DNAzyme was created "in vitro".
using the common DNA amplification technique called PCR or, the polymerase-chain-reaction.
Compared to RNA 
    The DNAzyme can be coded in longer sequences and with higher level of purity.
The longer sequences were proven, by Santoro and Joyce in 1998 to have a higher catalytic efficiency.
Created in vitro by Stephen W. Santoro and Gerald F. Joyce  

7. 10-23 is highly selective, cleaving only the purine-pyrimidine bond
Cleavage
GCGUGGGU
AGAGAGAGG
l l l l l l l l
l l l l l l l l
CGC AC CC A G G C T A G C T A C A A C G A
CTC T C TC C
 The DNAzyme operates by binding an RNA substrate at two specific substrate-recognition domains, which can be modified according to the substrate in question.  The DNAzyme then cleaves the phosphodiester linkage between a purine and a pyrimidine.
Notice how the DNA forms a loop when cleaving the RNA.  This loop is important in the enzyme structure and is a result of the divalent ion causing the DNA to fold upon itself.
10-23 is highly selective, cleaving only the purine-pyrimidine bond

8. Mechanism: Structural vs. Chemical
Tertiary interaction
Charge density
Ex.
Ribozymes HH, HP, and VS with monovalent ions
HDV ineffective
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However, studies done with the hammerhead, hairpin, Hepatitis Delta Virus, and VS ribozymes, which all normally function with a divalent metal ion, showed that these ribozymes also worked effectively in the presence of high concentrations of monovalent cations, including Lithium, Sodium, and Ammonium.  This suggests the charge of the cofactors may be the major contributing factor that aids in catalysis, folding the tertiary structure of the nucleic acids into a conformation conducive to RNA cleavage.
This hypothesis was further supported by the success of high hydrostatic pressure in producing the same results as the monovalent ions did, forcing the structure of the enzyme-substrate complex into a certain conformation and leading to catalysis and reaction.

9. Mechanism: Structural vs. Chemical
Santoro-Joyce mechanism models
Stephen Santoro and Gerald Joyce, who labeled DNAzyme 10-23, proposed two main mechanisms through which divalent metal ions aided the cleavage of phosphodiester bonds of RNA.  
The two mechanisms work very similarly, with each forcing the 2' hydroxyl to donate an oxygen atom to the phosphate, destabilizing the backbone of RNA and cleaving the RNA into two pieces.
Mechanism A suggests the divalent cation induces hydroxide ions in solution to deprotonate the 2' hydroxl, releasing the oxygen atom to destabilize the phosphate molecule.
Mechanism B proposes that the divalent metal cation instead acts as a Lewis acid.  A Lewis base will donate an electron pair to the metal ion, and then deprotonate the 2' hydroxyl, leading to an end pathway similar to Mechanism A.
One piece of evidence supporting the idea that the mechanism of the divalent metal ions is directly chemical in nature is the failure of the trivalent ion Cobalt (III) hexamine to aid cleavage, meaning that greater charge density does not always lead to cleavage.
Pubs.acs.org.ezproxy.drew.edu/doi/pdf/ /bi ?cookieSet=1

10. Divalent Ions vs. Monovalent Ions
Divalents
Monovalents
The effectiveness is already recognized
Still no consensus whether structural or chemical
Could prove that reaction only requires a concentration of positive charge

11. HYPOTHESIS monovalent cations
If are effective in operating as a cofactor for DNAzyme 10-23,
then the function of metal ion cofactors is
rather than in nature.
structural
chemical
Based on the success of monovalent ions in acting as cofactors in RNAzyme catalyzed reactions, we hypothesized that monovalent ions Lithium, Sodium, and Ammonium will prove just as effective in acting as cofactors in DNAzyme catalyzed reactions, demonstrating that the function of metal ion cofactors in nucleic acid catalysis is structural in nature.

12. Materials and Methods Change buffer

13. Materials and Methods

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

23. Questions?