What is the effect of DNA concentration on total fluorescence intensity?

Beautiful data. We saw low anisotropy, in absence of binding agent, when using plastic multi-well trays and this went away when the plastic was pre-blocked with a solution containing BSA.

Article DNA Binding Affinity and Sequence Permutation Preference of ...

Hi

I am surprised to see the trend. 

The first point (no DNA) is well justified. A free small molecule should exhibit lower anisotropy value compared to its bound state. Hence the black point (no DNA) is obtained at the lower anisotropy value than the first white circle.

Remaining part of the plot (with increasing DNA concentration) does not explain the binding. As more DNA is added to the solution (if I assume final [DNA] is at least twice than the free [dye]) the equilibrium should shift to the bound state, which should result a higher anisotropy for each dye-DNA complexation step. So I would expect a mirror image curve of what I see. 

Based on the data shown here I doubt about any binding interaction between the dye and the DNA.

It appears to me that first addition of the DNA initiates a dyes aggregation process and the aggregated dyes break the aggregation at higher [DNA]. 

Moreover it could be from other reasons, like change in pH/ionic strength. Is this a double stranded DNA? Does the DNA addition change the pH/ionic strength of the medium?

Thank you everyone for your suggestions and ideas. We'll make some more experiments changing the ionic strength and adding competitive polyanions (ssDNA), which would be somewhat the same idea as the BSA blocking described by Martin... thanks!!!

By the way, intensity emission changes quite a bit (almost double) in the first addition of DNA, but then it changes very little throughout the rest of the titration (slight increase). So, to me it looks like initial charge-induced aggregation, followed by formation of the right complex as more sites are available.

According to your explanation and data, the anisotropy of the small molecule when bound to DNA at a low small molecule:DNA stoichiometry is slightly lower than the anisotropy of the unbound small molecule. This is rather surprising.

Do you plan to do a titration in a lower range of DNA concentrations to capture the initial rise?

Will try to do that, but it's a very steep increase... will keep you posted. yes, i thought about the lower anisotropy... not happy about it either. Thanks Adam

Wow this is some interesting data, and I feel a steady-state anisotropy measurement may not be sufficient to tease out what is going on.

Let me first echo Abhigyan and Adam's surprise at the data. The initial low data point is expected. Small molecules rotate quickly, depolarize quickly, and therefore show lower anisotropy.

The second point about the fluorescence intensity is important too. The intensity increase is indicating a longer fluorescence lifetime. What's weird is that a longer lifetime would cause the anisotropy to decrease given the same rotational correlation time (see Perrin Eq 10.44, link below).

It is possible that the longer lifetime is coming not from the loss of quenching, but excited states crossing. Binding to DNA may cause the dye to stabilize a different excited state with a different r0. It would explain the sudden increase in anisotropy and intensity. You could prove that to yourself by making your solution very viscous with glycerol or sucrose. Prepare the solution in unbound and bound forms and then add sucrose. This will grind rotation to a halt, report r0, and tell you if it has changed.

Now why does anisotropy decrease as the [DNA] increases? Well it could be physical if the DNA prefers a more compact configuration in the presence of more DNA, but that doesn't feel very good (I have no experience with DNA!). I think it's more likely that you are looking at a changing ratio of different dye states (e.g. 1- different excited states, 2- difference aggregation states, 3- free solution state. r_total = a*r1 + b*r2 + c*r3 ; total dye = a + b + c)

Steady state anisotropy is a bugger to interpret, and big assumptions about the dye have to be made. If you have access to a fluorescence lifetime system, I think you'll have better luck. Seeing how the number of lifetimes change with the addition of DNA (e.g. 1 to 2+) will tell you how many dye species are present, and a rotational correlation time can be extracted from the data with a decent anisotropy fitting model. How that correlation time changes with additional DNA will give you your binding information.

Good luck and tell us what you learn!

https://books.google.com/books?id=-PSybuLNxcAC&pg=PA367&lpg=PA367&dq=perrin+equation&source=bl&ots=xesYmCtlLV&sig=s6ADispF-Vuja9PkjXjLyxuW47g&hl=en&sa=X&ved=0ahUKEwiegaqdr6LWAhWO2YMKHQGqCagQ6AEIWzAJ#v=onepage&q=perrin%20equation&f=false