Non-specific Effects of Morpholinos

[Jon D. Moulton]

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Good day, zebrafish folks;

If anyone has more information on non-specific effects of Morpholinos, 
plese post your replies to the list -- those of us at Gene Tools want to 
know about these as well!

One interesting report we heard is that filter sterilization of all 
materials can decrease these effects, though be careful as Morpholinos 
will associate with some filter materials, leading to  decreased
Morpholino concentrations.  We found some Pall Acrodisc filters have 
little affinity for Morpholinos.

An interesting strategy to avoid toxicity of unusually toxic sequences  is
to use two translation blocking Morpholinos targeting the same 
transcript, for instance a 5'-UTR oligo and a start codon oligo.  Since 
many groups already have two such oligos for specificity confirmation,  it
is a small step to inject them in combination at decreased
concentrations.  Investigators have reported to us that toxic effects 
have been eliminated while preserving specific knockdown by using this 
technique.

An excellent investigation of a catastrophic off-target interaction 
appeared in the sea urchin literature and is available online:
Coffman JA, Dickey-Sims C, Haug JS, McCarthy JJ, Robertson AJ.
Evaluation of developmental phenotypes produced by morpholino antisense 
targeting of a sea urchin Runx gene. BMC Biol. 2004 May 07;2(1):6.
http://www.pubmedcentral.gov/picrender.fcgi?tool=pmcentrez&blobtype=pdf&artid=419381
I expect that similar events can occur in zebrafish, though for that 
organism I have not seen such detailed documentation of off-target 
interactions.

When Morpholinos are designed by Gene Tools, we DO NOT BLAST the
sequences to check for potential interactions with other transcripts.  We
suggest that the best strategy for ordering oligos is to submit  sequences
for our free design service, BLAST the complements of the  oligos to check
for potential off-target interactions, and then specify  the exact
antisense sequence when you order the oligos.  Ordering by  specifying a
GenBank number or cDNA sequence means you won't have the  opportunity to
do a BLAST search.

I usually suggest that the BLAST homology should be below about 80%, but 
that cutoff % is fairly arbitrary.  15 contiguous bases of homology is 
the minimum inactivating length for a Morpholino (that is, less than a 
15-mer oligo won't block translation), but if you flank 10 bases of 
homology with a mispair at either side and then add some homologous  bases
at the outside borders, you can still get a knockdown.  Five  mispairs
spread throughout a 25-mer almost always gives
loss-of-knockdown (so we sell five-mispair oligos as specificity
controls).  If you place all five mispairs in one end of the oligo, you 
still get 20 contiguous complementary bases in a 25-mer and those 20 
bases would retain considerable antisense activity.  My point is that 
when you find a partially-homologous region, following a rule of thumb 
like "less than 80% homology is OK" can lead to trouble -- you still  need
to look at distribution of the mispairs.

Another problem is that BLAST searches will not tell you if you have 12 
bases of homology, 1 mismatch, and 12 more bases of homology.  BLAST  will
simply report this as 12 bases of homology, but it is really a 
single-mismatch that would very likely lead to strong knockdown by the 
Morpholino.  A search tool that finds these cases would be useful for 
checking the likelihood of off-target interactions by Morpholinos.

Here are a few more factors to consider when comparing mismatched 
sequences: losing a C-G pair impacts the oligo activity more than losing 
an A-T pair (three H-bonds for G-C compared to two for A-T) and watch  for
forming G-T pairs, which are non-Watson-Crick pairing but still form  two
hydrogen bonds.

Thanks for sharing your experiences with off-target effects of
Morpholinos.  The more everyone understands the experimental
characteristics of the oligos, the better tool they become.

Regards,

   - Jon

      Jon D. Moulton, Ph.D.
      GENE TOOLS, LLC
      jmoulton at gene-tools.com
      www.gene-tools.com
      (541) 929-7840 x1201


SKucenas wrote:
> --------------------------------------------------------------------------
>
> I'm currently using morpholinos to characterize the function of my
protein of interest.  I've read a lot about non-specific effects of MOs
and I know what toxicity looks like with this technology.  Besides the
routine neurodegeneration and slowed development, has anyone come
> across other non-specific effects?  In particular, pigmentation or
neural alterations that occur without any other signs of toxicity?  Any
information would be greatly appreciated.  Thanks!
>
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>
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