t1: the effects of metal cations on 10-23 dnazyme activity by: anvesh annadanam, raghunandan avula,...
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T1: The effects of metal cations on 10-23 DNAzyme activity By: Anvesh Annadanam, Raghunandan Avula, Nathan Buchbinder, Jonathan Chen,
Sarah Cuneo, Ruth Fong, Michael Granovetter, Paul Lee, Tyler Nisonoff, Renuka Reddy, Meera Trivedi, Han-Wei Wu
Advisor: Dr. Adam G. Cassano
Assistant: Jyotsna Ramachandran
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The Big ThreeDNA – Deoxyribonucleic AcidRNA – Ribonucleic AcidProteins
http://www.scq.ubc.ca/a-monks-flourishing-garden-the-basics-of-molecular-biology-explained/
http://ias-cnsh.org/CNSH%C4%90%E1%BB%99ngV%E1%BA%ADt/CNSHTrongTh%C3%BAY/tabid/60/BlogDate/2009-02-28/DateType/month/Default.aspx
http://www.mpsciences.com/index-2.html
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Which Came First?DNA Proteins RNA
http://library.thinkquest.org/C0122429/intro/genetics.htm
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Ribozymes RNA is capable of enzyme activities
in certain casesFunctions are vital to lifeShows a mixture of functions
between DNA and enzymes (proteins)
Newest discovery: DNAzyme
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DNAzymesDiscovered in 1997DNA with enzymatic functionsCapable of site-specific cleavage of
RNA molecules
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10-23 DNAzymeCleaves target RNA in a sequence specific manner
Requirement:Metal cations as cofactors
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10-23 DNAzyme: Previous Research
Mg2+ cation worksMonovalent cations not as efficient
Other salts not nearly as effective
Properties of cofactors in reaction still not fully understoodRole of cofactors in the reaction
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Medical Applications RNA retrovirusesTreatment can be potentially damaging
DNAzymes show promise
http://www.deanza.fhda.edu/workforceed/images/06%20medical%20lab.jpg
http://www.mydogella.com/steth.jpg
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Purpose of Our ProjectFunctional mechanism of the 10-23
DNAzyme and its cofactorsLigand exchangeIonic Radius
Charge of the Mg2+
http://img.search.com/thumb/f/fd/CoA6Cl3.png/200px-CoA6Cl3.pngSantoro SF and Joyce GW 1998
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Questions to be AnsweredWhich factor is most important?
Ligand exchangeMolecular size
Which concentration works the best?
http://www.made-in-china.com/showroom/starchemicalssellers/product-detailAMjmsnZgaGpz/China-Strontium-Carbonate.html
http://cvbinasejahtera.blogspot.com/2009/09/bahan-bahan-kimia-industri.html
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ReactionsTotal Volume 10 µL
10-23 DNAzyme 1 µLRNA Substrate 1 µL EPPS Buffer 1 µL EDTA (ethylene diamine tetra-acetic
acid) 1 µL Monovalent Salts (NaCl) 1 µLCobalt Hexamine/ Strontium Chloride
+ H20 = 5 µL
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15% Polyacrylamide PAGE Gel with Urea
http://biotech.matcmadison.edu/resources/proteins/labManual/images/220_04_039.gif
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Mig
ratio
n of
DN
A
p. 2-12
+
-
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Controls and Standards
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Initial Cobalt and Strontium Reactions
Reactions ran for one hourTrial one gel ran for 45 minTrial two gel ran for one hourSYBR green staining time increase
Reactions at 37 oC
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Cobalt Hexamine: Initial Trials
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Strontium: Initial Trials
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Experimental Improvements 1.Reaction time increased
2.Gel Electrophoresis running time increased
3.SYBR-Green staining time increased
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Strontium: Trial 3
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Strontium Trial 3: Percent of RNA Product After Electrophoresis
10mM 50mM 100mM 150mM 250mM Positive Control0
10
20
30
40
50
60
70
80
90
Reaction Solution
% P
rod
uct
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Cobalt Hexamine: Trial 3
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Cobalt Hexamine Trial 3:Percent of RNA Product after Electrophoresis
RNA Sub
stra
te
Prod
uct S
tand
ard
Negat
ive
Contro
l
Posit
ive
Contro
l
130m
m
100m
m
75m
m
50m
m
25m
m
0
10
20
30
40
50
60
70
80
90
Reaction Solution
% P
rod
uct
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ConclusionsStrontium propertiesCobalt Hexamine propertiesLigand exchange is more important than size
Concentration effects
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AcknowledgementsDr. Adam Cassano
(Break a leg)Dr. David MiyamotoDr. Paul QuinnJohn and Laura
OverdeckBayer HealthCareBristol-Myers SquibbJewish Communal FundThe Ena Zucchi Trust Johnson & Johnson
RocheNovartisThe Crimmins Family
Charitable Foundation
Independent College Fund of New Jersey
The Edward W. and Stella C. Van Houten Memorial Fund
Drew University
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Questions?