Another Quenching Method -Static Quenching- 2004. 4. 20 Lim Hee Woong

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

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 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.

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 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 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