Another Quenching Method-Static Quenching-
2004. 4. 20Lim Hee Woong
Quenching Dynamic 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
Static Quenching vs. Dynamic Quenching
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
Hypothetical Heterodimer
Experimental Result Fluorophore and quencher
Cy3.5, FAM BHQ1
(Black hole quencher 1)
Dual labeld
Fluorophore Only
BHQ only(unscaled)
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)
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
Relation between fluorescence and concentration Linear relationship Intra rather intermolecular quenching
Some other results… See reference
Discussion Advantage
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.
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?
Reference M. K. Johansson et al.
Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probesJ. Am. Chem. Soc. 2002, 124, 6950
M. K. Johansson et al.Intramolecular dimers: a new design strategy for fluorescence-quenched probesChem. Eur. J. 2003, 9, 3466
http://www.biosearchtech.com/ Black hole quencher
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