1. Another Quenching Method -Static Quenching-
Lim Hee Woong

2. Quenching Dynamic Quenching Static Quenching In the excited state
Förster quenching
Falling off at a rate of 1/R6
Dexter quenching
Depends on the spatial overlap of orbitals
Static Quenching
In the ground state

3. Static Quenching vs. Dynamic Quenching

4. Different from FRET in that…
No excitation and intramolecular dimer
Donor and quencher moieties bind together to form ground state complex
Stemless molecular beacon
Depends on the overlap of absorption spectra
FRET depends on the overlap of absorption and emission spectra

5. Hypothetical Heterodimer

6. Experimental Result Fluorophore and quencher Cy3.5, FAM
BHQ1 (Black hole quencher 1)

7. Dual labeld
Fluorophore Only
BHQ only (unscaled)

8. Absorption spectra of the probes
More overlap of absorption spectra in Cy3.5
Not equal with the sum of each spectrum
Dual labeled
Fluorophore Only
BHQ only (unscaled)

9. Fluorescence spectra of the probes
Fluorescence spectra changed though BHQ is dark quencher.
More quenching in Cy3.5 in despite of less overlap of absorption and emission spectra
More overlap of absorption spectra
Dual labeled
Fluorophore Only

10. Relation between fluorescence and concentration
Linear relationship
Intra rather intermolecular quenching

11. Some other results…
See reference

12. Discussion Advantage But! No need to consider stem loop
No constraint in emission-absorption spectral overlap
Make it easy to design probe
But!
Thermal stability of fluorophore-quencher ground state complex
Most likely to be significant only in room temperature.

13. Application to Current Project
Probe with intramolecular dimer
In the weighted encoding method with FRET
No need to eliminate unbound probe
Consideration
Annealing temperature?
Appropriate pairs of fluorophore and quencher?
Enough Förster distance?

14. Reference
M. K. Johansson et al. Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes J. Am. Chem. Soc. 2002, 124, 6950
M. K. Johansson et al. Intramolecular dimers: a new design strategy for fluorescence-quenched probes Chem. Eur. J. 2003, 9, 3466
Black hole quencher

15. Another Consideration in Fluorimetry
Fluorescence signal
Relation between signal intensity and concentration
Fluorescence signal in case of mixed fluorescence
FRET and distance
Distance dependence of FRET especially in DNA structure