peker milas, mark arsenault ben gamari, richard buckman, lori goldner 24 november 2010 single...

Peker Milas, Mark Arsenault Ben Gamari, Richard Buckman, Lori Goldner

24 November 2010

Single Molecule FRET Measurements on A Model RNA, Cy3, Cy5 complex

FRET Mechanism

https://netfiles.uiuc.edu/tjha/www/newTechnique.html

Details (1)

ANn

JQR

45

206

0 128

10ln9

6

0

1

1

RR

E R: Distance between fluorophoresR0: Forster distance, i.e. where the energy transfer is 50%

Q0: Fluorescence quantum yield of donor in the presence of acceptor: Dipole orientation factor: Some nice irrational and transcendental numberNA: Avogadro’s numbern: Refractive index of mediumJ: Spectral overlap integral

dfJ AD4 : Wavelength

fD: Normalized donor emission spectrum as a function of wavelength: Acceptor molar extinction coefficient

http://www.microscopyu.com/tutorials/flash/spectralprofiles/index.html

Details (2)

Details (3)

2 3

2 3

A D

Energy of interac

1 ˆ ˆE

tion

lectric

ˆ

field of D: 3

1

of μ in E

: ˆ3

D D D

AD D Dia AAE

E R Rn R

En R

R R

http://online.physics.uiuc.edu/courses/phys598OS/fall05/lectures/FREThistory.ppt

An example FRET measurement on the surface

• A double stranded 16mer RNA• Cy3 and Cy5 are attached on opposite sites• Both are connected to the RNA at 5’ position• Dye+RNA complex attached to the glass surface by biotin/streptavidin

Cartoon View Streptavidin/Biotin

Streptavidin

Streptavidin/Biotin

300

250

200

150

100

50

0

300250200150100500

1 μm

300

250

200

150

100

50

0

300250200150100500

1 μm

Emission of Cy3

Emission of Cy5

Crosstalk Realization

Same FRET measurement in a water droplet without Streptavidin/Biotin

FRET-labeled molecule

Conclusion

• Observed FRET efficiency is different in both cases• The best candidates are geometrical effects and charge distributions

Droplet Making

http://www.detergentsandsoaps.com/emulsifiers.html

http://commons.wikimedia.org/wiki/File:Surfactant.jpg

First attempts

1. Fluorinated oils FC-70 and FC-772. Surfactant Trition X-1003. Ultrasound for creating shear on the mixture

Problems

1. High O2 concentration in oil2. Possibility of having multiple emulsions3. Buoyancy (droplet drift in observation volume)

21

Triton X-100

hydrophilic tail

hydrophobichead

polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether

FC-70

perfluoropentylamine[a.k.a. perfluoroamylamine]

fluorineatoms

FC-772-(nonafluorobutyl)heptafluorofuran

After Many Trials…

1. Silicone oil (Polydimethylsiloxane or PDMS)2. A grafted nonionic silicone surfactant

(Poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propyl]methylsiloxane])3. Stirring in a beaker, application of ultrasound etc…

http://en.wikipedia.org/wiki/Polydimethylsiloxane

http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=480320|ALDRICH&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC

http://www.dowcorning.com/content/discover/discoverchem/forms-mixtures-surfactants.aspx

Particle Tracking System

MD Simulations

Orientation depndence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

A. Iqbal,S.Arslan, B. Okumus, T. J. Wilson, G. Giraud, D. G. NormanT. J. Ha D. M. Lilley, PNAS 2008

Let’s remember our fluorophores

Cy3 Cy5

3prime

5prime

3prime 5ns

5prime 5ns

D.A. Case, T.A. Darden, T.E. Cheatham, III, C.L. Simmerling, J. Wang, R.E. Duke, R. Luo, R.C. Walker, W. Zhang, K.M. Merz, B.P. Roberts, B. Wang, S. Hayik, A. Roitberg, G. Seabra, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, X. Wu, S.R. Brozell, T. Steinbrecher, H. Gohlke, Q. Cai, X. Ye, J. Wang, M.-J. Hsieh, G. Cui, D.R. Roe, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, T. Luchko, S. Gusarov, A. Kovalenko and P.A. Kollman (2010), AMBER 11, University of California, San Francisco.

Alex A. Granovsky, Firefly version 7.1.G, www http://classic.chem.msu.su/gran/firefly/index.html, PCGAMESS/FIREFLY N. M. O'Boyle, A. L. Tenderholt and K. M. Langner. J. Comp. Chem., 2008, 29, 839-845.

AcknowledgementsAcknowledgements

Former Group Members

Mark ArsenaultJames MooreAlex KiriakopoulosRose CitronSilvia IonescuChristophe WilhelmRyan D’souza

Funding

NSF MCB award 0920139

UMass start-up funding

NIST internal funding

PI

Prof. Lori Goldner

Group Members

Dianwen Zhang

Ben Gamari

Richard Buckman

Collaborators

Brent Krueger (Hope College)

Thank you…