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Shrinking mRNA Peaks over time in CE

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jeffmoonchop:
Hi all, I'm testing the stability of mRNA by CE over time. The mRNA is encapsulated in lipid nanoparticles so when I prepare the samples I break down the particles to release the mRNA using Triton. When I only just make the particles the mRNA purity is high at the same as the standard (usually around 93%), but over time the purity decreases as expected especially at 25C conditions. For example, it reduces to 50% after three months.

The thing is by normal degradation wouldn't you expect other peaks to appear if the mRNA strand is hydrolysing or breaking up?

What I see is no extra peaks but the peak area/height just reduces. Before the peak there is always a front or hump, That front doesn't change but because the peak height changes the ratio of peak areas reduces compared to the front.

My question is, what is happening to the mRNA? Is it just evaporating or is it being bound more strongly by the lipid over time so it's not being released? Does anyone have an idea and how I could test it to confirm what is happening?

many thanks

Babcock_Hall:
RNA is subject to a number of degradation reactions, including transphosphorylation.  Ronald Breaker has studied transphosphorylation.  I am at a loss to explain why other peaks do not show up.

Yggdrasil:
RNA can be hydrolyzed down to single nucleotides, which likely are too small to be seen in the capillary electrophoresis (e.g. it looks like the LM marker corresponds to 15 nt).  Furthermore, if the nucleotides are dephosphorylated to nucleosides, they would not be seen by electrophoresis since they do not have any charge.  Single nucleotides or small oligonucleotides may also not be detected by the CE instrument depending on the detection method used (e.g. CE instruments commonly use fluorescent dyes that bind to nucleic acids, and these dyes will not efficiently bind single nucleotides or very short oligonucleotides).

jeffmoonchop:
great thanks for that answer. I'll look more into those degradation mechanisms. I'm not a biologist so its great help.

jeffmoonchop:
Would you be able to offer a way to stabilise mRNA to prevent degradation? I've looked at a few papers and they all seem to be enzyme based but I'm treating the mRNA like an API, are there any chemical additives I could use?

I'm planning on using antioxidants such as vit C and E in combination to scavenge oxygen radicals. Do you have any other ideas?

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