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Questions about Morpholino Oligo Design


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#1 rustyshackleford

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Posted 16 March 2011 - 02:37 PM

Hey Everyone,

I was wondering how many nucleotides upstream from the start codon (i.e. in the leader sequence) are needed in order to design a good morpholino oligo to target a gene of interest? Can a successful morpholino oligo begin at the start codon itself?

Unfortunately, the genome for my model organism has not been sequenced, so I had to clone the sequence for my gene of interest myself. Using RACE-PCR, I was able to sequence about 80 nucleotides upstream from the start codon; can I assume that these nucleotides are part of the leader sequence, since they occur directly before the start codon? I'm not sure that these upstream nucleotides are meaningful and that I can reliably use that part of the sequence for morpholino design.

Can anyone offer any advice?

Many thanks in advance!

--Rusty

#2 Jon Moulton

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Posted 17 March 2011 - 06:50 AM

Hi Rusty,

You can target from the start codon downstream (covering the AUG). You can target upstream in the 5'-UTR. If you have sequence upstream of the start codon, that's the 5'-UTR (leader sequence). Here are some notes on targeting translation blockers.

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** Targetable region for translation blocking

For blocking cap-dependent translation, a Morpholino can target anywhere between the 5' cap and the start codon and can extend downstream into the coding sequence as long as the start codon is covered. To see why this works, consider the steps at the beginning of translation. A group of proteins and the small ribosomal subunit bind at the 5' cap and then other initiation factors bind, forming the initiation complex. The initiation complex scans through the UTR to the start codon. At the start codon the large subunit binds, the initiation factors dissociate and translation proceeds through the coding region.

If we can get in the way of the initiation complex by binding a Morpholino to the UTR we can prevent the initiation complex from reaching the start codon, but once the large subunit binds and forms an entire ribosome then a Morpholino oligo cannot stop its progression; the ribosome just displaces the downstream oligo from the mRNA and reads through. This is why the targetable region for translation blocking extends from the 5' cap to the start + 25 bases.

There are two reasons why we prefer to target at the start. First, the quality of sequencing deposited in public databases is often poor in the UTR, especially for older sequences. More than once we've found cloning vector sequence reported in UTR. Second, though rare in vertebrate genomes, internal ribosome entry sites (IRES) do exist and can allow a ribosome to "short-circuit" a Morpholino-blocked target. So, when selecting oligo sequences, I like to start at the start codon and analyze the possible oligos in that region, then if necessary I'll work upstream into the UTR until I find a good target.

For results of targeting some actual translation-blocking oligos, see figure 1 on the page:
http://www.gene-tools.com/node/18

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Let me know how I can help.

- Jon
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com




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