Morpholino antisense oligomers: the case for an RNase H-independent structural type.
TL;DR: In cell-free and cultured-cell systems where one wishes to block the translation of a messenger RNA coding for a normal protein, RNase H-independent morpholino antisense oligos provide complete resistance to nucleases, generally good targeting predictability, generally high in-cell efficacy, excellent sequence specificity, and very preliminary results suggest they may exhibit little non-antisense activity.
About: This article is published in Biochimica et Biophysica Acta.The article was published on 1999-12-10. It has received 689 citations till now. The article focuses on the topics: RNase P & RNase H.
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TL;DR: It is shown here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish, and conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebra fish embryos.
Abstract: The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos.
2,582 citations
Additional excerpts
...We show here that antisense, morpholino-modified oligonucleotide...
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TL;DR: The molecular mechanisms by which antisense oligonucleotides can be designed to modulate RNA function in mammalian cells and how synthetic oligon nucleotides behave in the body are focused on.
Abstract: Dramatic advances in understanding of the roles RNA plays in normal health and disease have greatly expanded over the past 10 years and have made it clear that scientists are only beginning to comprehend the biology of RNAs. It is likely that RNA will become an increasingly important target for therapeutic intervention; therefore, it is important to develop strategies for therapeutically modulating RNA function. Antisense oligonucleotides are perhaps the most direct therapeutic strategy to approach RNA. Antisense oligonucleotides are designed to bind to the target RNA by well-characterized Watson-Crick base pairing, and once bound to the target RNA, modulate its function through a variety of postbinding events. This review focuses on the molecular mechanisms by which antisense oligonucleotides can be designed to modulate RNA function in mammalian cells and how synthetic oligonucleotides behave in the body.
1,153 citations
TL;DR: It is shown here that MO off-targeting results in induction of a p53-dependent cell death pathway, and p53 inhibition could potentially be applicable to other systems to suppress off- target effects caused by other knockdown technologies.
Abstract: Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.
1,019 citations
Cites background from "Morpholino antisense oligomers: the..."
...Morpholino phosphorodiamidate oligonucleotides (MOs) [1] and short inhibitory RNAs (siRNAs) [2] have been instrumental to induce sequence-specific gene knockdown in multiple systems....
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TL;DR: It is discussed how the use of morpholinos can lead to misleading results, including off-target effects, and controls that will allow researchers to interpret morpholino experiments correctly are suggested.
Abstract: One of the most significant problems facing developmental biologists who do not work on an organism with well-developed genetics - and even for some who do - is how to inhibit the action of a gene of interest during development so as to learn about its normal biological function. A widely adopted approach is to use antisense technologies, and especially morpholino antisense oligonucleotides. In this article, we review the use of such reagents and present examples of how they have provided insights into developmental mechanisms. We also discuss how the use of morpholinos can lead to misleading results, including off-target effects, and we suggest controls that will allow researchers to interpret morpholino experiments correctly.
645 citations
Cites background from "Morpholino antisense oligomers: the..."
...In these cases, the antisense reagent interfered with gene function by hybridising to endogenous RNAs and by mediating their degradation via RNaseH (Summerton, 1999)....
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...MOs do not act through an RNaseH mechanism but instead can be designed to inhibit translation (Summerton, 1999) (Fig....
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TL;DR: The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.
596 citations
Cites background from "Morpholino antisense oligomers: the..."
...In cell-free translation experiments, they were shown to have greater efficiency and specificity than other antisense oligos, and tests showed excellent solubility and stability characteristics (Summerton, 1999)....
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References
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TL;DR: The potent immune activation by CpG oligon nucleotides has impli-cations for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.
Abstract: Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.
3,742 citations
"Morpholino antisense oligomers: the..." refers background in this paper
...In addition, S-DNAs containing the sequence PuPu-C-G-Py-Py have been shown to trigger B cell activation [11] and S-DNAs containing four or more contiguous guanines have been shown to form a tetrameric complex which can cause a variety of nonantisense e¡ects [12]....
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TL;DR: It is reported here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane, suggesting an energy-independent mechanism of translocation not involving classical endocytosis.
2,149 citations
"Morpholino antisense oligomers: the..." refers background in this paper
...They subsequently discovered that an amphiphilic 16 amino acid sequence in that protein is largely responsible for this transmembrane transport activity [53]....
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TL;DR: An overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells.
Abstract: Antisense promised major advances in treating a broad range of intractable diseases, but in recent years progress has been stymied by technical problems, most notably inadequate specificity, ineffective delivery into the proper subcellular compartment, and unpredictable activity within cells. Herein is an overview of the design, preparation, and properties of Morpholino oligos, a novel antisense structural type that solves the sequence specificity problem and provides high and predictable activity in cells. Morpholino oligos also exhibit little or no nonantisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs.
1,253 citations
TL;DR: It is shown that the HSV-1 structural protein VP22 has the remarkable property of intercellular transport, which is so efficient that following expression in a subpopulation the protein spreads to every cell in a monolayer, where it concentrates in the nucleus and binds chromatin.
1,113 citations
"Morpholino antisense oligomers: the..." refers background in this paper
...Several other groups have also identi¢ed proteins which are excreted and then appear to pass directly across the plasma membrane into recipient cells ^ including VP22 from herpes simplex virus [54] and galaparan [55]....
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TL;DR: The use of a previously described 17-mer phosphorothioate for structure-function analysis of 2'-sugar modifications and the results demonstrate the importance of target affinity in the action of antisense oligonucleotides and of RNase H as a mechanism by which these compounds exert their effects.
804 citations
"Morpholino antisense oligomers: the..." refers background in this paper
...E¤cacy can be increased even further by going to a chimeric oligo wherein the weakerbinding RNase H-competent segment of the chimera is bounded on one or both sides by a higher-a¤nity stretch of derivatized RNA [34]....
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