T1: The effects of metal cations on 10-23 DNAzyme activity By: Anvesh Annadanam, Raghunandan Avula,...

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

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 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

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

rod

uct

Cobalt Hexamine: Trial 3

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

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?