Eugen Uhlmann

PNA: Synthetic Polyamide Nucleic Acids with Unusual Binding Properties

The astonishing discovery that peptide nucleic acids (PNAs, B=nucleobase), in spite of their drastic structural difference to natural DNA, are better nucleic acid mimetics than many other oligonucleotides has resulted in an explosion of research into this class of compounds. The synthesis, physical properties, and biological interactions of PNAs as well as their chimeras with DNA and RNA are summarized here.

The synthesis of polyamide nucleic acids using a novel monomethoxytrityl protecting-group strategy

Abstract The preparation of novel monomethoxytrityl (Mmt) protected monomers for the synthesis of polyamide nucleic acids (PNAs) is described. The use of base-labile acyl-type nucleobase protecting groups and of a succinyl-linked solid-support offers a synthetic strategy similar to standard oligonucleotide synthesis conditions. This strategy has been successfully applied for the synthesis of PNAs of mixed base sequence.

Novel synthetic routes to PNA monomers and PNA-DNA linker molecules

Abstract Novel methods for the preparation of monomethoxytrityl (Mmt) protected aminoethylglycine building blocks and dimethoxytrityl (Dmt) protected hydroxyethylglycine derivatives useful for the synthesis of polyamide nucleic acids (PNAs) and PNA/DNA chimeras are described. The protecting group strategy employed for PNA monomer synthesis produces easily isolable intermediates, minimizes chromatographic purification, and is suitable for large-scale monomer synthesis.

Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression

Abstract The design and use of minimally modified oligonucleotides for specific inhibition of gene expression is discussed. The “minimal” protection strategy is a combination of the end-capping technique and the protection of internal pyrimidine positions which are the major sites of endonuclease degradation. By reducing the number of phosphorothioate modifications needed to make the oligonucleotide resistant to nuclease degradation, non-sequence-specific effects, which are frequently observed with uniformly phosphorothioate-modified oligonucleotides, can be reduced.

PNAs: synthetische Polyamidnucleinsäuren mit außergewöhnlichen Bindungseigenschaften

Auserst uberraschend war die Erkenntnis, das Peptidnucleinsauren (PNAs, B=Nucleobase) trotz ihrer drastisch vom naturlichen DNA-Ruckgrat abweichenden Struktur besser als die meisten Oligonucleotidderivate als Nucleinsauremimetica genutzt werden konnen. Die Synthese, physikalischen Eigenschaften und biologischen Wechselwirkungen sowohl der PNAs als auch ihrer Chimaren mit DNA und RNA werden hier zusammenfassend beschrieben.

Oligodeoxynucleotide Targeted to the αv Gene Inhibits αv Integrin Synthesis, Impairs Osteoclast Function, and Activates Intracellular Signals to Apoptosis

The αv integrin subunit is highly expressed in osteoclasts where it dimerizes with β1 and β3 subunits to form receptors for vitronectin and bone sialoproteins. Inhibition of osteoclast adhesion and function has previously been achieved by αvβ3 antibodies or Arg‐Gly‐Asp–containing peptides which have the disadvantages of blocking a single receptor type, or of being rather nonspecific, respectively. Here we show that αv integrin expression in rabbit osteoclasts can be inhibited by partially phosphorothioated antisense oligodeoxynucleotide (ODN) spanning the adenine‐uracil‐guanine (AUG) translational start site of the human/rabbit αv gene, a procedure which offers the advantage of affecting all the αv receptors with high efficiency. The αv antisense ODN caused a dose‐dependent, substrate‐specific reduction of osteoclast adhesion and bone resorption. Control ODNs, such as sense, inverted, and mismatch, were without effect, providing evidence of specificity of the antisense reagent. It is likely as a consequence of loss of substrate interaction, the antisense ODN induced osteoclast retraction and apoptosis, increase of the cyclin/cyclin‐dependent kinase complex inhibitor p21WAF1/CIP1, and inhibition of the cell survival gene, bcl‐2. Although the expression of the cell death–promoting gene, bax, remained unchanged, a reduction of the bcl‐2/bax ratio, known to underlie the intracellular signal to apoptosis, was observed. This finding led us to hypothesize that these changes could provide a link between reduction of αv synthesis and osteoclast programmed death. In conclusion, this study provides novel insights into the use of αv antisense ODN as an efficacious mechanism for blocking osteoclast function and underscores for the first time the involvement of integrins in bone cell apoptosis. In vivo studies may verify potential application of this ODN as alternative therapy for bone diseases.

Synthesis of polyamide nucleic acids (PNAs) using a novel Fmoc/Mmt protecting-group combination

Abstract The preparation of 9-Fluorenylmethoxycarbonyl(Fmoc) protected building blocks for the synthesis of polyamide nucleic acids (PNAs) is described. Use of 4-Methoxyphenyldiphenylmethyl (Mmt)-protecting groups for the exocyclic amino function of the nucleobases enhances the solubility of the monomers and allows final deprotection by mild acid treatment. The novel synthetic route is exemplified by the synthesis of heptameric and octameric PNAs.

Oligonucleotide technologies: synthesis, production, regulations and applications

The two-day meeting ‘Oligonucleotide technologies’, organised by IBC Global Conferences Limited and chaired by M Gait (Medical Research Council, UK), was the first event of this type in Europe. It covered a wide range of oligonucleotide-based approaches useful for the identification and validation of new therapeutic targets as well as for the creation of novel oligonucleotide-based drugs. Recent progress in the preclinical and clinical development of new oligonucleotide-based therapeutics as well as new platforms and systems for DNA microarrays, allowing massively parallel gene expression studies, were discussed. Furthermore, new approaches for overcoming challenges in the large scale synthesis and functionalisation of oligonucleotides were revealed. The following meeting highlights are focussed on contributions devoted to the development of novel oligonucleotide-based drugs, such as antisense oligonucleotides, immunostimulatory CpG oligonucleotides and spiegelmers. Drug candidates discussed here are related to various indications, including cancer, infectious diseases and asthma. Besides highlighting of specific clinical candidates, emphasis is put on the basic principles of oligonucleotide-based drugs and approaches towards the improvement of their properties.

Nuclease stability as dominant factor in the antiviral activity of oligonucleotides directed against HSV-1 IE110

The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.

Synthesis of Polyamide Nucleic Acids (Pnas), Pna /Dna-Chimeras and Phosphonic Ester Nucleic Acids (Phonas)

Abstract The synthesis of polyamide nucleic acids (PNAs) and derivatives thereof by different synthetic routes is described. The first strategy makes use of 9-Fluorenylmethoxycarbonyl (Fmoc)/monomethoxytrityl (Mmt) protected building blocks, whereas the second approach involves the use of Mmt/acyl protected monomers, which allows the preparation of PNADNA chimera. Additionally, a block coupling strategy is presented for the synthesis of novel phosphonic ester nucleic acids (PHONAs).