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?