An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers (original) (raw)
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Novel Antisense and Peptide Nucleic Acid Strategies for Controlling Gene Expression
Biochemistry, 2002
Antisense oligonucleotides have the potential to make revolutionary contributions to basic science and medicine. Oligonucleotides can bind mRNA and inhibit translation. Because they can be rapidly synthesized to be complementary to any sequence, they offer ideal tools for exploiting the massive amount of genome information now available. However, until recently, this potential was largely theoretical, and antisense experiments often produced inconclusive or misleading outcomes. This review will discuss the chemical and biological properties of some of the different types of oligomers now available and describe the challenges confronting in vitro and in vivo use of oligonucleotides. Oligomers with improved chemical properties, combined with advances in cell biology and success in clinical trials, are affording powerful new options for basic research, biotechnology, and medicine.
RNase H-independent antisense activity of oligonucleotide N34P54 phosphoramidates
Nucleic Acids Research - NAR
Oligonucleotide N34∀P54 phosphoramidates are a new and promising class of antisense agents. Here we report biological properties of phosphoramidate oligonucleo- tides targeted against the human T cell leukemia virus type-I Tax protein, the major transcriptional transactiva- tor of this human retrovirus. Isosequential phosphoro- thioate oligodeoxynucleotides and uniformly modified and chimeric phosphoramidate oligodeoxynucleotides containing six central phosphodiester linkages are all quite stable in cell nuclei. The uniformly modified anti-tax phosphoramidate oligodeoxynucleotide does not acti- vate nuclear RNase H, as was shown by RNase protection assay. In contrast, the chimeric phosphorami- date-phosphodiester oligodeoxynucleotide is an effi- cient activator of RNase H. The presence of one or two mismatched nucleotides in the phosphodiester portion of oligonucleotides affected this activation only negligi- bly. When introduced into tax-transformed fibroblasts ex vivo, only the un...
Nucleic Acids Research, 2002
Use of antisense oligonucleotides is a versatile strategy for achieving control of gene expression. Unfortunately, the interpretation of antisenseinduced phenotypes is sometimes dif®cult, and chemical modi®cations that improve the potency and speci®city of antisense action would be useful. The introduction of locked nucleic acid (LNA) bases into oligonucleotides confers exceptional improvement in binding af®nity, up to 10°C per substitution, making LNAs an exciting option for the optimization of antisense ef®cacy. Here we examine the rules governing antisense gene inhibition within cells by oligonucleotides that contain LNA bases. LNAcontaining oligomers were transfected into cells using cationic lipid and accumulated in the nucleus. We tested antisense gene inhibition by LNAs and LNA±DNA chimeras complementary to the 5¢untranslated region, the region surrounding the start codon and the coding region of mRNA, and identi®ed effective antisense agents targeted to each of these locations. Our data suggest that LNA bases can be used to develop antisense oligonucleotides and that their use is a versatile approach for ef®ciently inhibiting gene expression inside cells.
Antisense and Antigene Properties of Peptide Nucleic Acids
Science, 1992
Peptide nucleic acids (PNAs) are polyamide oligomers that can strand invade duplex DNA, causing displacement of one DNA strand and formation of a D-loop. Binding of either a T10 PNA or a mixed sequence 1 5-mer PNA to the transcribed strand of a G-free transcription cassette caused 90to 100 percent site-specific termination of pol 11 transcription elongation. When a T10 PNA was bound on the nontranscribed strand, site-specific inhibition never exceeded 50 percent. Binding of PNAs to RNA resulted in site-specific termination of both reverse transcription and in vitro translation, precisely at the position of the PNA. RNA heteroduplex. Nuclear microinjection of cells constitutively expressing SV40 large T antigen (T Ag) with either a 15-mer or 20-mer PNA targeted to the T Ag messenger RNA suppressed T Ag expression. This effect was specific in that there was no reduction in ,B-galactosidase expression from a coinjected expression vector and no inhibition of T Ag expression after microinjection of a 10-mer PNA.
Nucleic Acids Research, 2003
Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA±DNA±LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2¢-O-methyl RNA±DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA±DNA±LNA gapmers with four or ®ve LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1±green¯uorescent protein (GFP) expression. A speci®c inhibition was observed with an estimated IC 50 of 0.06 nM. An LNA gapmer was found to be the most ef®cient single-stranded antisense oligonucleotide, with an IC 50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC 50~7 0 nM). In contrast, the ef®ciency of a 2¢-O-methyl-modi®ed oligonucleotide (IC 50~2 20 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA±DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.
Potent and nontoxic antisense oligonucleotides containing locked nucleic acids
Proceedings of The National Academy of Sciences, 2000
Insufficient efficacy and͞or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a highaffinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA͞DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA͞DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA͞DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.
Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs
Nucleic acids research, 2001
The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE co...
Antisense Oligonucleotides, A Novel Developing Targeting Therapy
Antisense Therapy [Working Title]
Antisense oligonucleotides (ASOs) have been validated as therapeutic agents and an important tool in molecular biology. Indeed, ASOs are used either in vitro or in vivo to generate mRNA selective knockouts. They can be used for human therapy since ASOs can inhibit specifically target genes especially whose are difficult to target with small molecules inhibitors or neutralizing antibodies. However, despite their specificity and broadness of use, some practical obstacles remain unsolved in antisense pharmacology, such as insufficient stability due to nucleases degradation activity, and poor cellular delivery as a result of low cellular uptake difficult biological membrane crossing. Moreover, in many cases, potential off-target effects and immunostimulation are also part of the problems derived from their use. In this review, we will discuss ASOs, their chemistry, limitation of use, some solutions to increase stability, and finally some of their therapeutical application.
Biochimie, 1998
Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives arc currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. cJt-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of or-ON-and [-~-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras. © Soci6t6 franqaise de biochimie et biologic mol6culaire / Elsevier, Paris antisense / oligonucleotide / ct-anomcric DNA / RNase L / RNase H / 2,5A