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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…