[jeffmoonchop]

Hi all. mRNA is negatively charged due to the phosphodiester group joining the bases. Experimentally, I am finding that mRNA charge varies based on the pH of the buffer its in.

This was done by monitoring migration of mRNA through a gel at different pH. A higher pH migrating the fastest. My question is what is causing the differences in overall net charge of the mRNA?

The pKa of mRNA is very low, in the 2 range. Normally you get a steep dropoff of charge for a particular molecule, but as mRNA is a polymer, I'm wondering if at low pH, some (not all) of the P-O^- groups turn into P-OH. As the pH increases, these H+ gradually vacate the P-OH, increasing the net charge of the mRNA.

We are seeing a drastic difference between pH 4 and 6. So I'm guessing that its due to the lack of P-OH groups.

Does anyone have any thoughts about this?

thanks

[Babcock_Hall]

Verdolino V et al., J. Phys. Chem. B 2008, 112, 16860–16873
It would be surprising but not unprecedented for a pK_a that is ordinarily around 0.7 to be perturbed into the range of 4 to 6.  I would think about two factors.  The first is that the conformation is known to affect the mobility of duplex DNA, but I am not sure whether this has been demonstrated for ssRNA, which is considerably different.  The conformation of the RNA might change as a function of pH, possibly owing to the behavior of base triplets.  The second is that the bases have their own dissociation constants.  I did a very quick search, and I would hazard a guess that adenine and cytosine might be responsible.  These two points (conformation and the ionization of bases) might be related.

[Yggdrasil]

One thought is that the ionic strength of your electrophoresis buffer is different at the different pH values, which is could be the source of the change in electrophoretic mobility.

Another possibility is that there are RNA secondary structures that change the shape (and therefore mobility) of the RNA which are sensitive to changes in pH in the 4-6 range.

Your hypothesis could be tested by titrating an mRNA sample and observing whether there are equivalence points that would suggest ionizable groups with pKa between 4-6.

[jeffmoonchop]

Thanks or your replies. My evidence is not only based on gels, but also the rate at which the mRNA associates with a cationic lipid.

I've also thought about secondary structure and I plan to do a heating experiment with UV-vis, where we might be able to determine the extent of structure at different pH. It would be hard to tease apart whether fast migration is due to charge or structure, or both. Highly structured species may migrate faster than a non-structure species of the same charge.

One thought is that the ionic strength of your electrophoresis buffer is different at the different pH values, which is could be the source of the change in electrophoretic mobility.

I'm using the same concentration of buffer for these experiments, for example 100mM citrate at varied pH.

I have also thought about the behavior of individual bases, where some may prevent or allow protonation of the phosphate more than others.

Thanks for the tips.

[Babcock_Hall]

The ionic strength of citrate varies more than other species as a function of pH, owing to its multiple charges.  There was a paper many years ago in Methods in Enzymology about buffers having the same ionic strength at various pH values; the buffers were intended for use in obtaining pH-rate data for enzymes, but the details escape me.  I am not sure whether or not these buffers are suitable for electrophoresis.  With respect, I am not yet convinced by the association rate constants with cationic detergents.  Could the detergents be associating with charges on the bases?

I wonder whether or not P-31 NMR might be helpful, but it depends on the size of the molecule.  P-31 is a classic way to obtain dissociation constants.