Blogs

Watching dynamics of RNA expression - paper describing a visualization technique.

Movie 1: https://www.biorxiv.org/highwire/filestream/112058/field_highwire_adjunct_files/3/366468-4.mp4

Movie 1 from supplemental information: in this zebrafish embryo, DNA is stained with red fluorescence. Carboxyfluoresceinaed Morpholino oligos targeting dre-miR-430 emit visible green fluorescence when they reach sufficient localized concentration. In red you can watch condensation of chromosomes, mitosis, and loosening of the chromatin. After a few divisions you will see green dots appear where groups of miR430 genes are being transcribed and capturing fluorescent-labeled Morpholinos. Each nucleus contains two dots, the maternal and paternal chromosomes revealing the site of miR430 transcription. The green dots disappear as the red chromosomes condense out of the chromatin for mitosis and gene expression halts for division. This movie shows the early-to-mid blastula stages and the onset of zygotic transcription occurs at mid-blastula, so you don't see the green dots appear during the first few cell divisions; early on the cells are expressing maternal mRNAs that are already present in the egg. The onset of zygotic transcription is where the embryo begins to rely on its own genome.

The paper: https://www.biorxiv.org/content/early/2018/07/15/366468

Hadzhiev Y, Qureshi H, Wheatley L, Cooper L, Jasiulewicz A, Nguyen HV, Wragg J, Poovathumkadavil D, Conic S, Bajan S, Sik A, Hutvagner G, Tora L, Gambus A, Fossey JS, Mueller F. A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation. BioRXive. 2018;[Epub] doi:doi.org/10.1101/366468.

A researcher asked about the half-life of the Morpholino. This is much of my response.

The half-life of the molecule is not very useful for experiment planning. The oligos do not degrade (Hudziak RM et al. 1996, Youngblood DS et al. 2007), but their activity is temporarily lost when they bind to complementary RNA; that binding rate is what sets the trajectory of biological activity. Eventually the RNA will degrade off the oligo and release it, but the RNA footprint can be protected from nuclease activity by the Morpholino so this is a slow process, leading to a persistent background of activity well below experimental utility (but reported as weak splice-modifying activity over three months after a single dose in mice, Wells 2008). We typically see about four days of useful knockdown in cultured cells or systemically with a Vivo-Morpholino. Larger doses of Morpholino will persist longer, but the resulting higher oligo concentration in cells might lead to some off-target RNA interaction (and the dose of a Vivo-Morpholino will be limited by toxicity). The rate of new transcription of a particular RNA is an important factor for the duration of a Morpholino knockdown; slow transcription helps the oligo activity persist longer, while rapid transcription can swamp the oligo quickly, leading to a short knockdown. The turnover rate of the protein affects how soon you can detect the knockdown; the oligo might halt transcription, but the protein concentration decreases as a function of its degradation and in some cases can be fairly slow.

Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, Weller DD. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev 1996 Winter;6(4):267-72.

Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60.
(note the persistence of the signals corresponding to the mass of the bare oligo without peptide)

Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008 Jun;154(3):623-31. Epub 2008 Apr 21.

A prp1 Morpholino injected into zebrafish with mutated prp1 caused significant phenotype at a dose of 6 ng/embryo but had little effect at lower doses (see Supplemental Figure S13). This is a case where the Morpholino-in-mutant experiment not only detected an off-target interaction of the Morpholino but defined a dose where the effect suddenly became strong. Doses of prp1 Morpholino below that threshold produced phenotype at roughly the rate of the standard control oligo injections.

Leighton PLA, Kanyo R, Neil GJ, Pollock NM, Allison WT. Prion gene paralogs are dispensable for early zebrafish development but have non-additive roles in seizure susceptibility. J Biol Chem. 2018 Jun 14. pii: jbc.RA117.001171. doi: 10.1074/jbc.RA117.001171. [Epub ahead of print]
http://www.jbc.org/content/early/2018/06/14/jbc.RA117.001171.abstract

"Consensus guidelines for the use and interpretation of angiogenesis assays" contains a very good discussion of Morpholinos and specificity. Nowak-Sliwinska et al. extensively cite the Stainier et al. "Guidelines for Morpholino Use in Zebrafish". There is a theme in the Morpholino discussion in Nowak-Sliwinska et al.'s paper with which I disagree. They state "However, the best and generally accepted validation for any MO phenotype is confirmation of the same phenotype in a zebrafish genetic mutant." [1] Is this best, or does it exclude a valuable function of Morpholinos? Following Stainier et al., I advocate that performing a specificity control by using a targeting Morpholino in a null background for the same transcript is a better validation for the MO. In a mutant undergoing compensatory changes in gene expression, Morpholinos checked with that method which produce no additional observed effects (in a compensated background) might present with more extreme phenotypes when used in a wild-type background (in the absence of compensation) and reveal useful information about gene function which is obscured in the mutants. Later in their discussion Nowak-Sliwinska et al. do address the utility of morphants whose phenotypes diverge from their corresponding mutants:

"Fourth, recent work has shown that upregulation of related compensating gene family members can sometimes occur in genetic mutants (by mechanisms that are not yet clear), while this does not appear to take place in MOs-injected animals [277], arguably making MOs a better representation of targeted loss of gene function in these cases."

To accept that a Morpholino might be producing accurate transcript-specific information even when mutant and morphant phenotypes differ, a rigorous specificity control is needed. Stainier et al. write: "A decisive approach to determine the optimal sequence and dose of a MO that does not cause off-target effects is to inject the MO into embryos whose genome (and whose mother’s genome, for maternally expressed genes) has been edited so as to eliminate the MO-binding site or to eliminate the function of the target gene" [2]. Using this specificity control allows application of Morpholinos to probe gene functions which are concealed by compensation in some mutants. I argue it is a better validation than observing agreement of mutant and morphant phenotypes because it is applicable to a broader range of genes.

[1] Nowak-Sliwinska P, Alitalo K, Allen E, Anisimov A, Aplin AC, Auerbach R, Augustin HG, Bates DO, van Beijnum JR, Bender RHF, Bergers G, Bikfalvi A, Bischoff J, Böck BC, Brooks PC, Bussolino F, Cakir B, Carmeliet P, Castranova D, Cimpean AM, Cleaver O, Coukos G, Davis GE, De Palma M, Dimberg A, Dings RPM, Djonov V, Dudley AC, Dufton NP, Fendt SM, Ferrara N, Fruttiger M, Fukumura D, Ghesquière B, Gong Y, Griffin RJ, Harris AL, Hughes CCW, Hultgren NW, Iruela-Arispe ML, Irving M, Jain RK, Kalluri R, Kalucka J, Kerbel RS, Kitajewski J, Klaassen I, Kleinmann HK, Koolwijk P, Kuczynski E, Kwak BR, Marien K, Melero-Martin JM, Munn LL, Nicosia RF, Noel A, Nurro J, Olsson AK, Petrova TV, Pietras K, Pili R, Pollard JW, Post MJ, Quax PHA, Rabinovich GA, Raica M, Randi AM, Ribatti D, Ruegg C, Schlingemann RO, Schulte-Merker S, Smith LEH, Song JW, Stacker SA, Stalin J, Stratman AN, Van de Velde M, van Hinsbergh VWM, Vermeulen PB, Waltenberger J, Weinstein BM, Xin H, Yetkin-Arik B, Yla-Herttuala S, Yoder MC, Griffioen AW.
Consensus guidelines for the use and interpretation of angiogenesis assays.
Angiogenesis. 2018 May 15. doi: 10.1007/s10456-018-9613-x. [Epub ahead of print] Review.
https://link.springer.com/article/10.1007/s10456-018-9613-x

[2] Stainier DYR, Raz E, Lawson ND, Ekker SC, Burdine RD, Eisen JS, Ingham PW, Schulte-Merker S, Yelon D, Weinstein BM, Mullins MC, Wilson SW, Ramakrishnan L, Amacher SL, Neuhauss SCF, Meng A, Mochizuki N, Panula P, Moens CB. Guidelines for morpholino use in zebrafish. PLoS Genet. 2017 Oct 19;13(10):e1007000. doi: 10.1371/journal.pgen.1007000. eCollection 2017 Oct.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.10...

This is an older review (2008), but has a nice discussion of Morpholinos and controls. Photo-Morpholino and Vivo-Morpholino use in zebrafish are not addressed. The newer strategy of using Morpholinos in CRISPR mutants for specificity control is more recent than this paper, as is the understanding of gene compensation in mutants versus relatively uncompensated phenotypes from knockdowns triggered by Morpholino microinjections.

Skromne I, Prince VE.
Current perspectives in zebrafish reverse genetics: moving forward.
Dev Dyn. 2008 Apr;237(4):861-82. doi: 10.1002/dvdy.21484. Review.

https://onlinelibrary.wiley.com/doi/full/10.1002/dvdy.21484

Here is an example of an unexpected splice-modifying outcome. An oligo was targeted to i1e2, which would normally be expected to skip exon 2. Instead, the oligo caused retention of intron 1. This is the usual outcome of an e1i1 oligo, but not of an i1e2 oligo. Figure 5B shows the gel and the RNA map: https://www.cell.com/cms/attachment/2119254818/2091259359/gr5.jpg

Burns DT, Donkervoort S, Müller JS, Knierim E, Bharucha-Goebel D, Faqeih EA, Bell SK, AlFaifi AY, Monies D, Millan F, Retterer K, Dyack S, MacKay S, Morales-Gonzalez S, Giunta M, Munro B, Hudson G, Scavina M, Baker L, Massini TC, Lek M, Hu Y, Ezzo D, AlKuraya FS, Kang PB, Griffin H, Foley AR, Schuelke M, Horvath R, Bönnemann CG. Variants in EXOSC9 Disrupt the RNA Exosome and Result in Cerebellar Atrophy with Spinal Motor Neuronopathy. Am J Hum Genet. 2018 May 3;102(5):858-873. doi: 10.1016/j.ajhg.2018.03.011.

https://www.cell.com/ajhg/fulltext/S0002-9297(18)30100-9

This is an open-access review covering the mechanism of eukaryotic RNA splicing and diseases caused by mutations in regions affecting splicing.

Abramowicz A, Gos M. Splicing mutations in human genetic disorders: examples, detection, and confirmation. J Appl Genet. 2018. doi: 10.1007/s13353-018-0444-7
https://rd.springer.com/article/10.1007%2Fs13353-018-0444-7

Review article
Nan Y, Shang Y-J. Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral Compounds. Front Microbiol. 2018;9:750. doi:10.3389/fmicb.2018.00750
https://www.frontiersin.org/articles/10.3389/fmicb.2018.00750/full

It is always prudent to screen proposed oligo sequence for potential off-target RNA interactions using BLAST. Partial homologies between a Morpholino's inverse complement and an RNA which are reported by BLAST are often in regions of the RNA where binding a Morpholino is unlikely to alter gene expression. If there is significant homology in a region where binding a Morpholino is likely to alter a transcript's expression, have us design another oligo.

Whether a BLAST hit presents a potential off-target effect depends on where the hit is located on an RNA as well as the degree of sequence similarity. Different people have different risk tolerance and you'll have to judge whether the risk is appropriate for your experiment. First BLAST the oligo's target sequence and consider the degree and location of similar sequence prior to ordering an oligo synthesis. Then you will also need to do subsequent empirical testing to check for off-target RNA interactions. Selecting and performing a specificity control experiment is an important step during your Morpholino experiments; specificity controls may include using a second non-overlapping oligo, doing an mRNA rescue, or using the oligo in a mutant that is null for the RNA the Morpholino targets.

BLAST the invert complement of the Morpholino sequence (the oligo's target sequence) in order to find places where the Morpholino might bind to off-target mRNA. For a translation blocker, at least BLAST the transcript sequences. For a splice modifier, at least BLAST genomic sequence. It is possible that a sequence from a 5'-UTR might occur elsewhere near a splice junction, or that a splice junction sequence might occur in a 5'-UTR, so for the most thorough check for unexpected interaction with RNA it is best to BLAST each oligo target against both transcript and genomic sequence.

Remember when searching for homologous targets that Morpholinos will only block translation when targeted to the UTR or first 25 bases of coding sequence. Also, Morpholinos can affect splicing if targeted in introns near intron-exon boundaries. If the Morpholino has homology to an off-target mRNA outside of these limited regions, binding of the oligo to the mRNA is less likely to affect expression of the off-target mRNA (though blocking regulatory sequences [e.g. exonic splice enhancers, 3'-UTR miRNA targets, poly-A signal sequences, etc.] may affect expression).

I usually suggest that the fraction of homologous bases should be below about 80%, but that cutoff % ignores important considerations about the distribution of the mispairs through the oligo. We have long stated that 14 to 15 contiguous bases of homology is the minimum inactivating length for a Morpholino (that is, less than a 14-mer oligo won't efficiently block translation), but if you flank 10 bases of homology with a mispair at either side and then add some runs of homologous bases at the outside borders, you can still get a knockdown. High CG content can make shorter homologous sequences active, since CGs pair with greater stability than ATs; in general, homologous sequences with high predicted Tm should be avoided if they are in an area which might affect gene expression. Recent reports suggest that homologies with as little as 11 bases matched can have off-target effects (see: BLAST homology and specificity controls), though the 11-base sequence reported has an unusually high Tm (see: Paper on Morpholino specificity and CRISPR-MO combination). Five mispairs spread throughout a 25-mer almost always gives loss-of-knockdown (so we sell five-mispair oligos as specificity controls, though there are far better 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.

Watch for multiple regions of homology, for example 12 bases of homology, 1 mismatch, and 12 more bases of homology. If you are just looking for contiguous homology you would interpret this as 12 contiguous bases of homology, but it is really a single-mismatch 25-base target that would very likely lead to strong knockdown by the Morpholino if it is in a place where the bound Morpholino would alter gene expression.

Here are a few more factors to consider: 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.

The BLAST algorithm is not an exhaustive sequence search, so it is not surprising you see some differences between different BLASTs of the same sequence. Screening using several databases is best and even with the same genome build, they will often produce different results; it really depends how much of your time you make available for the screen. BLASTING a sequence can sometimes catch a problem, but it is an endless rabbit-hole to explore so you have to set a limit on how long you will search a given sequence. In the end, a good physical specificity control should tell you if the targeting oligo is working specifically. BLASTing just helps eliminate some oligos up-front that will not survive the specificity control challenge.

"Due to genetic compensation, phenotypically wild-type mutants can become refractive to morpholino-induced phenotypes, providing a critical test both for genetic compensation and for the specificity of morpholino phenotypes."

Balciunas D. Fish mutant, where is thy phenotype? PLoS Genet. 2018;14(2):e1007197.
https://doi.org/10.1371/journal.pgen.1007197