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Is it appropriate to use Stern-Volmer equation in Hoechst-DNA...
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Loren McCune
Is it appropriate to use Stern-Volmer equation in Hoechst-DNA...
The Stern-Volmer equation is not appropriate for a competitive binding situation. In this case, there are two competing, saturable binding equilibria, which should be described by two equilibrium dissociation constants, at a low ligand:DNA ratio. (At a high ratio, there could be interactions between binding sites on the DNA, making the analysis much more complicated.) One of the dissociation constants is for the Hoechst dye, which binds in the minor groove of the DNA. The other is for the competitor, which excludes binding of the Hoechst dye.
Since the fluorescence of Hoechst is dependent on DNA binding, you can measure the competition for binding by the decrease in Hoechst fluorescence due to its displacement from the DNA. Calculating the Kds of the two compounds when titrating both simultaneously is challenging because it involves solving a cubic equation, but there is a workaround. It is easy to measure the Kd of the Hoechst dye by a DNA titration at a fixed Hoechst concentration by measuring the increase in fluorescence. You can then set up a competition assay in which the DNA concentration is set equal to the Hoechst Kd and the Hoechst concentration is set far below its Kd. The concentration of competitor that reduces the Hoechst fluorescence by half (IC50) will then be twice the Kd of the competitor. This math assumes that the competitor Kd is substantially higher than the concentration of DNA binding sites.
The DNA concentration has to be stated in terms of molarity. The average molecular mass of a base pair is 660 g/mole, so you can convert mg/mL of DNA to molar concentration of base pairs.
I would not describe the competitor as a quencher unless it is found to actually quench the fluorescence of Hoechst when both are bound simultaneously. This could happen by resonance energy transfer, or by static quenching if they bind right next to each other, or by dynamic quenching if the competitor collides with DNA-bound Hoechst. Fluorescence lifetime measurements would be needed to explore these possibilities.
The Stern-Volmer equation is not appropriate for a competitive binding situation. In this case, there are two competing, saturable binding equilibria, which should be described by two equilibrium dissociation constants, at a low ligand:DNA ratio. (At a high ratio, there could be interactions between binding sites on the DNA, making the analysis much more complicated.) One of the dissociation constants is for the Hoechst dye, which binds in the minor groove of the DNA. The other is for the competitor, which excludes binding of the Hoechst dye.
Since the fluorescence of Hoechst is dependent on DNA binding, you can measure the competition for binding by the decrease in Hoechst fluorescence due to its displacement from the DNA. Calculating the Kds of the two compounds when titrating both simultaneously is challenging because it involves solving a cubic equation, but there is a workaround. It is easy to measure the Kd of the Hoechst dye by a DNA titration at a fixed Hoechst concentration by measuring the increase in fluorescence. You can then set up a competition assay in which the DNA concentration is set equal to the Hoechst Kd and the Hoechst concentration is set far below its Kd. The concentration of competitor that reduces the Hoechst fluorescence by half (IC50) will then be twice the Kd of the competitor. This math assumes that the competitor Kd is substantially higher than the concentration of DNA binding sites.
The DNA concentration has to be stated in terms of molarity. The average molecular mass of a base pair is 660 g/mole, so you can convert mg/mL of DNA to molar concentration of base pairs.
I would not describe the competitor as a quencher unless it is found to actually quench the fluorescence of Hoechst when both are bound simultaneously. This could happen by resonance energy transfer, or by static quenching if they bind right next to each other, or by dynamic quenching if the competitor collides with DNA-bound Hoechst. Fluorescence lifetime measurements would be needed to explore these possibilities.
The Stern-Volmer equation is not appropriate for a competitive binding situation. In this case, there are two competing, saturable binding equilibria, which should be described by two equilibrium dissociation constants, at a low ligand:DNA ratio. (At a high ratio, there could be interactions between binding sites on the DNA, making the analysis much more complicated.) One of the dissociation constants is for the Hoechst dye, which binds in the minor groove of the DNA. The other is for the competitor, which excludes binding of the Hoechst dye.
Since the fluorescence of Hoechst is dependent on DNA binding, you can measure the competition for binding by the decrease in Hoechst fluorescence due to its displacement from the DNA. Calculating the Kds of the two compounds when titrating both simultaneously is challenging because it involves solving a cubic equation, but there is a workaround. It is easy to measure the Kd of the Hoechst dye by a DNA titration at a fixed Hoechst concentration by measuring the increase in fluorescence. You can then set up a competition assay in which the DNA concentration is set equal to the Hoechst Kd and the Hoechst concentration is set far below its Kd. The concentration of competitor that reduces the Hoechst fluorescence by half (IC50) will then be twice the Kd of the competitor. This math assumes that the competitor Kd is substantially higher than the concentration of DNA binding sites.
The DNA concentration has to be stated in terms of molarity. The average molecular mass of a base pair is 660 g/mole, so you can convert mg/mL of DNA to molar concentration of base pairs.
I would not describe the competitor as a quencher unless it is found to actually quench the fluorescence of Hoechst when both are bound simultaneously. This could happen by resonance energy transfer, or by static quenching if they bind right next to each other, or by dynamic quenching if the competitor collides with DNA-bound Hoechst. Fluorescence lifetime measurements would be needed to explore these possibilities.
The Stern-Volmer equation is not appropriate for a competitive binding situation. In this case, there are two competing, saturable binding equilibria, which should be described by two equilibrium dissociation constants, at a low ligand:DNA ratio. (At a high ratio, there could be interactions between binding sites on the DNA, making the analysis much more complicated.) One of the dissociation constants is for the Hoechst dye, which binds in the minor groove of the DNA. The other is for the competitor, which excludes binding of the Hoechst dye.
Since the fluorescence of Hoechst is dependent on DNA binding, you can measure the competition for binding by the decrease in Hoechst fluorescence due to its displacement from the DNA. Calculating the Kds of the two compounds when titrating both simultaneously is challenging because it involves solving a cubic equation, but there is a workaround. It is easy to measure the Kd of the Hoechst dye by a DNA titration at a fixed Hoechst concentration by measuring the increase in fluorescence. You can then set up a competition assay in which the DNA concentration is set equal to the Hoechst Kd and the Hoechst concentration is set far below its Kd. The concentration of competitor that reduces the Hoechst fluorescence by half (IC50) will then be twice the Kd of the competitor. This math assumes that the competitor Kd is substantially higher than the concentration of DNA binding sites.
The DNA concentration has to be stated in terms of molarity. The average molecular mass of a base pair is 660 g/mole, so you can convert mg/mL of DNA to molar concentration of base pairs.
I would not describe the competitor as a quencher unless it is found to actually quench the fluorescence of Hoechst when both are bound simultaneously. This could happen by resonance energy transfer, or by static quenching if they bind right next to each other, or by dynamic quenching if the competitor collides with DNA-bound Hoechst. Fluorescence lifetime measurements would be needed to explore these possibilities.
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