When a quantum dot is attached to 20-25 basepair double strand DNA and later an intercalator is added within such system, how can we calculate FRET distance between the quantum dot and intercalator?
How can we calculate from the Fluorescence data generated from spectrofluorimetry?
The Forster theory is used to calculate the distance between donor and acceptor. It requires collecting the corrected emission spectrum of the donor, the absorbance spectrum of the acceptor, calculating the overlap integral between the 2 spectra, and measuring the quantum yield of the donor with and without the acceptor. The value of the orientation factor kappa-squared is typically assumed to be 2/3 for organic fluorophores that can move relatively freely, but this may not be true for quantum dots because of their large size. Also, the acceptor, once intercalated, will not rotate freely. Therefore you will have to calculate limits to kappa-squared, which will require polarization measurements. In the end, your distance determination will have wide bounds. An additional problem is that a quantum dot is large compared to the distance being measured, and also the intercalator can bind at multiple places along the DNA. At best, you will be able to calculate some sort of apparent average distance. I don't know if anyone has published a theory for this type of system.
Why do you need to measure the distance anyway? If you know the size of the quantum dot and the length of the DNA, the geometry of the system is already well-defined. If you want to use the Forster theory properly, for example to figure out where along the DNA the intercalator binds, consider switching to a small organic fluorophore covalently linked to one end of the DNA.
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