Another Quenching Method -Static Quenching

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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

Text of Another Quenching Method -Static Quenching

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

  • QuenchingDynamic QuenchingIn the excited stateFrster quenchingFalling off at a rate of 1/R6Dexter quenchingDepends on the spatial overlap of orbitalsStatic QuenchingIn the ground state

  • Static Quenching vs. Dynamic Quenching

  • Different from FRET in thatNo excitation and intramolecular dimerDonor and quencher moieties bind together to form ground state complexStemless molecular beaconDepends on the overlap of absorption spectraFRET depends on the overlap of absorption and emission spectra

  • Hypothetical Heterodimer

  • Experimental ResultFluorophore and quencherCy3.5, FAMBHQ1 (Black hole quencher 1)

  • Dual labeldFluorophore OnlyBHQ only(unscaled)

  • Absorption spectra of the probesMore overlap of absorption spectra in Cy3.5Not equal with the sum of each spectrum

  • Fluorescence spectra of the probesFluorescence spectra changed though BHQ is dark quencher.More quenching in Cy3.5 in despite of less overlap of absorption and emission spectraMore overlap of absorption spectra

  • Relation between fluorescence and concentrationLinear relationshipIntra rather intermolecular quenching

  • Some other resultsSee reference

  • DiscussionAdvantageNo need to consider stem loopNo constraint in emission-absorption spectral overlapMake it easy to design probeBut!Thermal stability of fluorophore-quencher ground state complexMost likely to be significant only in room temperature.

  • Application to Current ProjectProbe with intramolecular dimerIn the weighted encoding method with FRETNo need to eliminate unbound probeConsiderationAnnealing temperature?Appropriate pairs of fluorophore and quencher?Enough Frster distance?

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

  • Another Consideration in FluorimetryFluorescence signalRelation between signal intensity and concentrationFluorescence signal in case of mixed fluorescenceFRET and distanceDistance dependence of FRET especially in DNA structure