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