Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.
purposes. For clinical applications like the antisenseBased on the oligomer-chip technology, oligonucleo-strategy (reviewed in Ref. 2), gram or even kilogram tide arrays were synthesized directly on polypropyl-amounts of oligonucleotides are required, prompting ene sheets by a modified phosphoramidite chemistry efforts on an up-scaling of synthesis yields (e.g., 3). For using b-eliminating nucleobase-protecting groups in many applications in molecular biology, on the other combination with a succinate solid-phase linker. This hand, notably DNA characterization and screening by method decouples the oligonucleotide deprotection hybridization and enzymatic assays like the polymerfrom the support cleavage procedure, in contrast to ase chain reaction (PCR) and DNA sequencing, oligostandard phosphoramidite chemistry. In addition to mer quantities in the picomole range are usually an being reliable substrates for hybridization experi-adequate amount, but larger numbers of oligonucleoments, the arrays also serve as source for the isolation tides are needed. Again, this has led to procedures aimof individual oligonucleotides. Technically, this al-ing at the simultaneous production of different oligonulowed for a direct control of the quality of the arrayed cleotides in small quantities (e.g., 4, 5). quence of an unknown nucleic acid is reconstructed from its hybridization binding pattern on a matrix which contains a comprehensive set of short oligonucleotide sequences (e.g., all 65,536 octamers). Initiated by the combination of solid-phase technolDuring our work concerned with developments toogy and the phosphoramidite chemistry introduced by ward a practical application of the SBH technique, such Beaucage and Caruthers (1), there has been much prog-as the ordering and selection of DNA fragments suitress in the automation of DNA synthesis. Today, the able for SBH analysis (10, 11), for example, or the sepreparation of synthetic oligonucleotides needed in bio-quence-independent leveling of oligomer binding stalogical, biomedical, and physical applications is usually bilities (12), it was apparent to us that such oligomer executed with commercial, automated DNA synthe-chips by design would be an ideal tool not only for sizers. These machines produce oligonucleotides in the screening procedures but also for the synthesis of nanomole up to micromole range. However, over the past few years, there have been two diametrical tend-2 Abbreviations
Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.
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