peker milas, mark arsenault ben gamari, richard buckman, lori goldner 24 november 2010 single...
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![Page 1: Peker Milas, Mark Arsenault Ben Gamari, Richard Buckman, Lori Goldner 24 November 2010 Single Molecule FRET Measurements on A Model RNA, Cy3, Cy5 complex](https://reader036.vdocument.in/reader036/viewer/2022062806/5697bfad1a28abf838c9c0f4/html5/thumbnails/1.jpg)
Peker Milas, Mark Arsenault Ben Gamari, Richard Buckman, Lori Goldner
24 November 2010
Single Molecule FRET Measurements on A Model RNA, Cy3, Cy5 complex
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FRET Mechanism
https://netfiles.uiuc.edu/tjha/www/newTechnique.html
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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
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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)
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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
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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
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Cartoon View Streptavidin/Biotin
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Streptavidin
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Streptavidin/Biotin
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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
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Crosstalk Realization
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Same FRET measurement in a water droplet without Streptavidin/Biotin
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FRET-labeled molecule
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Conclusion
• Observed FRET efficiency is different in both cases• The best candidates are geometrical effects and charge distributions
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Droplet Making
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http://www.detergentsandsoaps.com/emulsifiers.html
http://commons.wikimedia.org/wiki/File:Surfactant.jpg
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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)
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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
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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…
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http://en.wikipedia.org/wiki/Polydimethylsiloxane
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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
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Particle Tracking System
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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
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Let’s remember our fluorophores
Cy3 Cy5
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3prime
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5prime
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3prime 5ns
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5prime 5ns
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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.
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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)
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Thank you…