Dear all,
Am studying protein-DNA interactions, in which ssDNA is labelled at 3'end with 6FAM(fluorophore). After incubating 0.5nM of this labelled DNA with increasing concentration of my protein of interest(.1-1micromolar), there is a decrease in fluorescence anisotropy values. Instead of expected increase in fluorescence anisotropy, i always get a decrease. Does anyone has any advice or experience? I have checked the nuclease contamination in my reagents and protein solution, its totally clean. Nuclease contamination was checked by radiactivity based EMSA assay which is a very sensitive technique to check DNA fragmentation if nuclease is present. I donot have much experience of Anisotropy. Any help in this regard will be highy appreciable
One possibility is that the FAM fluorophore is somewhat immobilized in the ssDNA when the protein is absent because of an interaction of the FAM fluorophore with one or more of the bases. When the oligo binds to the protein, the FAM fluorophore is excluded from this interaction, making it more mobile. Try putting the fluorophore at the 5' end instead, using a different fluorophore, or changing the ssDNA sequence near the fluorophore.
This paper may provide some perspective:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1305492/
One possibility is that the FAM fluorophore is somewhat immobilized in the ssDNA when the protein is absent because of an interaction of the FAM fluorophore with one or more of the bases. When the oligo binds to the protein, the FAM fluorophore is excluded from this interaction, making it more mobile. Try putting the fluorophore at the 5' end instead, using a different fluorophore, or changing the ssDNA sequence near the fluorophore.
This paper may provide some perspective:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1305492/
Dear Sir,
sorry, I can't help you since I would also expect an increase in fluorescence anisotropy.
Kind regards
Herbert Schneckenburger
I think that Shapiro's answer is the most probable explanation. In my opinion, another way to interpret the data would be to propose that the protein is acting as a bridge between different DNA molecules, thereby bringing the flourophores together. If the fluorophores are separated by a distance of less than 10 nm Forster energy transfer can take place from one fluorophore to another. This particular type of Forster Energy transfer is called homotransfer. Homotransfer (also known as energy migration) can decrease the anisotropy value, since in the presence of homotransfer the energy "hopes" from fluorophore to fluorophore before being emitted as a photon and therefore the initial polarization is lost. Nonetheless, I believe that Shapiro's explanation is by far the simplest and therefore the best possible explanation at this point.
I agree with Sergio. Adding the protein may induce aggregation of DNA which would make the chromophores closer and a decrease of FA should be observed due to homo-FRET.
I agree with Sergio and Ivan. Aggregation of DNA can be induce a decrease of anisotropy (homo-FRET between your DNA molecules)
You may have sort of energy quenching occurring...
To check this run a steady-state emission scan and compare the steady-state emission spectra. If the fluorescence intensity also goes down as you increase the concentration of protein then there is some more evidence for sort of energy transfer.
If you can also measure the fluorescence lifetime of the fluorophore in increasing presence of the protein you should see the lifetime decrease.
You may wish to repeat the experiment using a longer linker to the fluorophore (keeping it further away from the fluorophore when bound and decreasing the chances of energy transfer).
I suspect that Adam's idea is likely what is happening for you, however I will propose an alternative hypothesis:
Anisotropy is a measure of rotational diffusion (https://en.wikipedia.org/wiki/Rotational_diffusion and also http://www.sciencedirect.com/science/article/pii/S0069804008702553) and the effect that it has on the polarization state of light. Rotational diffusion is effected by a number of things, but in your case you are likely concerned with sphere size and radius. The faster something rotates in solution the more of an effect it has on the polarization of light (and you see a low value for anisotropy), the slower something tumbles the less of an effect it has on anisotropy (so you see a larger value). Rotational diffusion can also be effected by the compaction of your substrate (effectively changing the radius of your rotating sphere). Since ssDNA has no defined linear structure in solution (like dsDNA would) it is possible that the addition of your protein of interest results in the formation (perhaps it induces a bend) of a uniform complex that is significantly more compact then the ssDNA was alone (the more compact structure would potentially have a smaller radius of rotation) and could lead to a lower anisotropic value.
@ Robert J. Bauer.
Yes it is known that my protein of interst leads to the bending of ssDNA and dsDNA. The idea seems too good to be true with my experiment. But please throw some light on the idea of bending and so low anisotropy values. If protein is making the DNA bend or kink and so low anisotropy but after formation of protein plus bent DNA complex, the anisotropy will be governed by Protein bound to DNA (which leads to low anisotropy) not by compacted or bent DNA. The protein is still in the solution in an equilibrium.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry