t1: the effects of metal cations on 10-23 dnazyme activity
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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 - PowerPoint PPT PresentationTRANSCRIPT
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. CassanoAssistant: Jyotsna Ramachandran
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
Which Came First?DNA Proteins RNA
http://library.thinkquest.org/C0122429/intro/genetics.htm
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
DNAzymesDiscovered in 1997DNA with enzymatic functionsCapable of site-specific cleavage of
RNA molecules
10-23 DNAzymeCleaves target RNA in a sequence specific manner
Requirement:Metal cations as cofactors
10-23 DNAzyme: Previous ResearchMg2+ 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
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
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
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
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
15% Polyacrylamide PAGE Gel with Urea
http://biotech.matcmadison.edu/resources/proteins/labManual/images/220_04_039.gif
Mig
ratio
n of
DN
A
p. 2-12
+
-
Controls and Standards
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
Cobalt Hexamine: Initial Trials
Strontium: Initial Trials
Experimental Improvements 1.Reaction time increased
2.Gel Electrophoresis running time increased
3.SYBR-Green staining time increased
Strontium: Trial 3
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
rodu
ct
Cobalt Hexamine: Trial 3
Cobalt Hexamine Trial 3:Percent of RNA Product after Electrophoresis
RNA Subst
rate
Produ
ct Sta
ndard
Negati
ve Con
trol
Positi
ve Con
trol
130m
m10
0mm
75mm
50mm
25mm
0102030405060708090
Reaction Solution
% P
rodu
ct
ConclusionsStrontium propertiesCobalt Hexamine propertiesLigand exchange is more important than size
Concentration effects
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
Questions?