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Antisense DNA And Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press COMMENTARY Antisense DNA and RNA: progress and prospects Using antisense DNA or RNA fragments to block the School of Medicine), to enhance the activity of oligonu­ expression of selected genes, and thereby assess their cleotide methylphosphonates, have prepared derivatives function, is a powerful new tool for the molecular biolo­ that are further modified with psoralen. Irradiation with gist. It is an approach that promises to be particularly 365-nm light results in irreversible cross-linking of the useful in higher eukaryotes, where the genetic tools ap­ oligonucleotide to the target sequence. Such derivatives plicable to yeast and bacteria are not available. There are block protein synthesis very effectively—in in vitro also obvious therapeutic implications in the use of translation systems inhibition occurs in the micromolar antisense inhibition to block the expression of targeted concentration range. Claude Helene (INSERM) described viral genes or oncogenes. Last December a conference at a similar approach in which iron and copper chelate the Banbury Center of Cold Spring Harbor Laboratory complexes were covalently attached to oligonucleotides. explored these aspects of antisense RNA and DNA. In the presence of reducing agents and oxygen, these A number of years before antisense RNA became fash­ complexes generate hydroxyl radicals that cleave the ionable, Paul Zamecnik and Mary Stephenson demon­ complementary strand to which the oligonucleotide is strated the feasibility of using short antisense oligodeoxy- hybridized. This approach has many interesting applica­ nucleotides to block the expression of targeted genes tions in vitro. Its use in vivo, though, requires a mechan­ within intact cells (Zamecnik and Stephenson 1978). ism to activate the system only after hybridization of This work provided the first evidence that oligonucleo­ the oligonucleotide to the targeted message. tides, although highly negatively charged, could be Helene also described studies of a new class of oligo­ transported into cells at some finite rate. John Good- nucleotides built from nucleosides having the a-ano- child (Worcester Foundation) described electron micros­ meric configuration rather than the naturally occurring copy studies using radiolabeled derivatives that provided p-form. These oligonucleotides hybridize strongly with further evidence for intracellular uptake of oligonucleo­ complementary sequences. Strands of a-(3 hybrids run tides. With added evidence from other laboratories (Holt parallel, in contrast to the antiparallel orientation of et al. 1988; Wickstrom et al. 1988), it is clear that oligo­ P-p duplexes. The a-anomers are highly resistant to nu­ nucleotides can be taken up into mammalian cells and clease degradation. However, they do not form sub­ can exert inhibitory effects on selected genes. The gener­ strates with mRNAs that are recognized by RNase H ality of this phenomenon and the mechanism of trans­ and, therefore, are much less effective inhibitors than port, however, remain uncertain. normal oligonucleotides. To facilitate intracellular transport of oligonucleo­ Degradation of oligonucleotides in mammalian cells tides, Paul Miller and Paul T'so and their colleagues in­ and in blood occurs most rapidly by exonucleases that vestigated analogs in which the negatively charged can be blocked by end-group modifications (Walder). Un­ phosphate groups of the DNA strand are replaced by fortunately, in Xenopus eggs, a system in which anti- neutral methylphosphonate links. Intracellular trans­ sense inhibition would be extremely useful for develop­ port of these derivatives is enhanced, but their intrinsic mental studies, endonucleases are also very abundant. activity is far less than that of unmodified oligonucleo­ Oligonucleotides blocked at both ends are degraded rap­ tides. The concentration of oligonucleotide methylphos- idly after microinjection (Doug Melton, Harvard Univer­ phonates required to inhibit protein synthesis in vitro is sity). Degradation of the targeted message is observed at generally between 100 and 400 fxM, compared with high concentrations of the oligonucleotide {Xenopus 0.5-5 )ULM for the corresponding unmodified sequences. eggs are very rich in RNase H), but the injection of the The important difference is that oligonucleotide meth- required amount of oligonucleotide is toxic to the em­ ylphosphonates do not form substrates for RNase H (J. bryo. Walder and R. Walder, University of Iowa). To dissect Why not use antisense RNA in the Xenopus system? out the contribution of RNase H, poly(rA)-oligo(dT) was Although RNA : RNA duplexes do not form substrates used to block the activity of the enzyme present in retic­ for RNase H, hybridization of a long antisense RNA se­ ulocyte lysates. This approach demonstrated that the ar­ quence to the 5'-end of a mRNA may block translation rest of translation by oligodeoxynucleotides which hy­ directly (Melton 1985). Unfortunately, antisense RNAs bridize within the coding region or over the initiation do not work well in Xenopus eggs either. The discovery codon is dependent on cleavage of the targeted mRNA of a helix unwinding activity in Xenopus eggs provided a by RNase H. In some cases, but not all, binding of anti- cogent explanation for this result (Bass and Weintraub sense sequences to the 5' end of mRNAs was found to 1987; Regbagliati and Melton 1987), but the problem may inhibit protein synthesis directly, presumably by inter­ be more complex. Harold Weintraub (Fred Hutchinson fering with the initiation of translation (see also, Lawson Cancer Research Center) presented evidence that this et al. 1986). factor not only unwinds RNA : RNA duplexes but also Miller and co-workers (Johns Hopkins University modifies the RNA strands in such a manner that they 502 GENES & DEVELOPMENT 2:502-504 © 1988 by Cold Spring Harbor Laboratory ISSN 0890-9369/88 $1.00 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press are unable to rehybridize. It would seem unlikely that remains capable of hybridization over the target se­ such an extensively modified mRNA would still be quence. Perhaps in this case, hybridization blocks translatable. If that supposition proves to be true, the normal translation termination. unwinding activity would not account for the un­ Many other examples demonstrating the utility of an­ masking of maternal mRNAs during differentiation, as tisense DNA and RNA were presented at this meeting. initially suggested, or the failure of antisense RNAs to Space permits the mention of only a few. All trypano- arrest translation of targeted mRNAs in Xenopus eggs. some mRNAs contain a common leader sequence of 35 Whatever their function, such unwinding factors appear nucleotides at their 5' end (Walder et al. 1986; Corne- to be widely distributed. Richard Wagner and Kazuko Hssen et al. 1986). Jean-Jacques Toulme (INSERM) re­ Nishikura (Wistar Institute) reported the presence of a ported that a nonanucleotide complementary to a por­ similar unwinding activity in HeLa cells and in five tion of this sequence, and containing an acridine deriva­ other mammalian cell lines. In mouse 3T3 fibroblasts, tive attached to the 3' end of the molecule, killed T. the activity is expressed in a cell cycle-specific manner. brucei cells in culture. Although high concentrations of The level of expression is very low in cells arrested in the oligonucleotide were required (80 fxM), the effect was quiescence and it increases sharply upon induction of shown to be sequence specific. The acridine group is es­ proliferation with fetal calf serum. sential for activity. Whether this is due to facilitated up­ Modification of RNA by the unwinding factors found take or decreased degradation of the oligonucleotide, or in Xenopus and other systems interferes with its detec­ to increased stability of the DNA : RNA duplex due to tion on Northern blots, and may lead to the erroneous intercalation of the dye, is unknown. conclusion that the targeted message has been degraded. Randy Moon (University of Washington School of To determine if degradation actually has occurred, the Medicine) described the use of antisense RNA to inhibit message must be probed for outside of the region com­ the expression of the cytoskeletal protein 4.1 during em­ plementary to the antisense RNA. bryonic development in Xenopus laevis. As in the Recently, inhibition of expression of the X cll gene by studies described above, microinjection of the antisense endogenous antisense RNA has been shown to be depen­ RNA into Xenopus eggs was ineffective. However, mi­ dent on cleavage of the mRNA by RNase III (Krinke and croinjection of a plasmid bearing the antisense sequence Wulff 1987). RNase III cleaves both the sense and the an­ flanked by the Moloney sarcoma virus long terminal re­ tisense strands in the RNA : RNA duplex. The target of peat to provide for a high level of expression proved to be the antisense RNA is at the 3' end of the message. The very successful. By the gastrula stage, antisense tran­ transposase gene of the insertion element IS 10 is also scripts became detectable in poly(A)'^ RNA, and, there­ regulated post-transcriptionally by antisense RNA after, cytoplasmic levels of the endogenous 4.1 tran­ (Nancy Kleckner, Harvard University). In this case, the script decreased dramatically. Kinetic studies suggest target sequence lies at the 5' end of the
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