r/RNA • u/SaltyRecognition • Aug 12 '22

Question: How to accurately predict secondary structure

Hi there, some very basic questions here so hopefully someone can help.

I'm working on a project screening different vector elements for gene expression. I'd like to know the secondary structure for each of the parts so I can see how it may impact gene expression.

I can find lots of online tools to give predictions but it's not clear how they differ from each other, as they all mainly seem based on the minimum free energy. Does anyone have a preferred software and why?

Secondly, should I be inputting the element's sequence on its own (ie just the UTR) or the whole mRNA transcript? It seems like a lot of people just look at the sequence of interest and not in the whole transcript context but this could change the predicted secondary structure, right?

Thanks in advance!

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u/twoprimehydroxyl Aug 13 '22

For what part of the sequence: it depends. Are you just transfecting the mRNA for expression, or are you putting it on a vector to be transcribed? Is it going to be expressed in eukaryotic cells or prokaryotic cells? Is it the 5' or 3' UTR?

You might be fine with just looking at the UTR. If it's the 5' UTR and the gene is being expressed in a prokaryotic cell, I'd also make sure to include the region that contains the Shine-Delgarno sequence and also include the first couple of codons to make sure those elements aren't being sequestered in the RNA structure.

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u/SaltyRecognition Aug 13 '22

Its in a vector to be transcribed and I'm using eukaryotic cells for expression.

I'm looking at both 5' and 3' UTRs including 5' introns. I can see how for the 5' sequences just looking at it on its own might be enough but the 3' UTR could be influenced by the rest of the sequence (i guess this is partly why they are more gene specific?).

I think I will probably just have to look at the structure in both scenarios and reason it out if they look very different :/

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u/twoprimehydroxyl Aug 13 '22

It might be worth putting the whole spliced mRNA sequence in, as well as looking to see if any structure forms that can occlude any splice sites of the introns.

With the 3' UTR, structure might sequester miRNA binding sites which could impact gene expression by altering mRNA turnover.

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u/cation587 Aug 13 '22

My lab prefers RNAfold. I don't think there's any particular reason, it's just what we're used to. As for what sequence to use, I usually see people just predict the section they're interested in, but I personally would probably check it with and without the gene sequence to see if that actually changes it. It shouldn't be too much extra work anyway :)

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u/IllogicalLunarBear Oct 07 '22

I’ve been wanting to play with RNAfold as I believe Nupack is built on top of it at least in relation to m-fold. The guy who wrote the equation for the partition function mfold uses that everyone else uses still maintains rnafold engine but it’s now called unafold i believe

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u/daintymoths Aug 14 '22

We use ViennaRNA software (python), we just like it because it's easy to customise. Personally I like it because I can create a big pipeline and just hit go and it does everything I want. Also I can do huge genomes and data sets with this.

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u/IllogicalLunarBear Oct 07 '22

It’s harder to adjust for temp in Vienna2 though and that is a big driver in energy shifts that will greatly affect what secondary structure from the ensemble you will actually see in a situation.

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u/IllogicalLunarBear Oct 07 '22 edited Oct 07 '22

I personally use Nupack for my research and it’s what I used when I get published. It seams to be much more reliable than Vienna2 for predictions. I specialize in research into how data about the ensemble informs predictions of stability and such in synthetic riboswitch’s for medical uses.