Bridged nucleic acids reloaded

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (20 -O,40 -aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.Fil: Soler Bistue, Alfonso J. C.. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Tolmasky, Marcelo E.. California State University; Estados Unido

Development of photosensitiser conjugates for furan-oxidation based interstrand crosslinking to nucleic acids

Esta tesis tiene embargado el acceso al texto completo hasta el 22-03-2019Tesis doctoral inédita cotutelada por la Universiteit Gent y la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Orgánica. Fecha de lectura: 22-09-201

Chemical synthesis and enzymatic incorporation of artificial nucleotides

Deutsche Übersetzung des Titels: Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotide

From cell penetrating peptides to peptoids and polyamines as novel artificial molecular transporters

In recent years, RNA interference has gained a lot of importance as a tool for posttranscriptional silencing of genes due to its high specificity, efficiency and ease of application. In mammalian cells RNAi is triggered by the application of 21 bp short dsRNAs, so-called short interfering RNAs (siRNAs). Numerous studies indicate the great potential of RNAi in the therapy of viral infections and inherited diseases. Cell-penetrating peptides can be used to apply siRNAs to primary cells, non-dividing cells or fully grown organisms, that are diffcult to transfect. These short positively charged peptides are internalized by cells. They can be detected in the endosomes, lysosomes but also in the cytosol. If attached to CPPs, large cargo molecules can be taken up with a high efficiency that surpasses that of most conventional transfection methods. In the work presented here, peptide-coupled siRNAs (pepsiRNAs) have been developed as a novel tool for transient RNAi in mammalian cells. The peptides were attached to the siRNA by a disulfide bond, that is cleaved under the reducing conditions of the cytosol and thus releases the siRNA cargo. PepsiRNAs are readily taken up by many cell types that are difficult to address by conventional transfection methods. An siRNA-induced downregulation of the targeted genes was observed at concentrations between 10 and 100 nM. SiRNAs with a 5´-thiol modification upon their sense-strand were generated by in vitro transcription, for which a thiol-modified nucleotide was synthesized via an optimized route. The cell-penetrating peptides Penetratin and Tat were recombinantly expressed as fusion-proteins with glutathione-S-transferase (GST) and purified with a modified procedure to overcome the strong membrane interaction of the GST-tagged CPPs. Recombinant TEV protease was expressed to cleave the CPPs from the fusion tag, and the cleavage activity was assessed by comparison with commercially obtained TEV protease. Thus, alternative routes to the building blocks for pepsiRNAs have been provided to scale up the amount of pepsiRNAs. Finally, small molecules with cell-penetrating properties have been developed as a future replacement of the peptide moiety. Fluorescently labeled peptoids of differing length with amine-functionalized side chains have been shown to enter different mammalian cells lines at concentrations in the lower micromolar range by an endocytosis dependent mechanism. Cationic molecules as small as spermine attached to fluorescein are taken up by an endocytosis-related mechanism. By the same approach porphyrins were delivered into the interior of the cells, where they exhibited a cytotoxic effect upon illumination, so that spermine-coupled porphyrins may be developed into a novel drug for photodynamic therapy.Entwicklung neuer Delivery-Strategien für siRNAs - Von zellpenetrierenden Peptiden zu Peptoiden und Polyaminen als neuartige molekulare Transporter In den vergangenen Jahren, hat die RNA Interferenz (RNAi) aufgrund ihrer Spezifität, Effizienz und einfachen Anwendbarkeit eine große Bedeutung als Technik für das posttranskriptionale Gene-Silencing gewonnen. In Säugerzellen wird RNAi durch die Gabe von 21 bp kurzen doppelsträngigen RNAs, sogenannten short interfering RNAs (siRNAs), ausgelöst. Zahlreiche Studien belegen das große Potential dieser Methode für die Therapie viraler Infektionen und Erbkrankheiten. Um siRNAs auch in schlecht transfizierbare Systeme wie primäre und nicht-teilende Zellen bzw. ausgewachsene Organismen einzubringen, können zellpenetrierende Peptide (CPPs) verwendet werden. Diese kurzen positiv geladenen Peptide werden von Zellen aufgenommen, wo sie in Endosomen, Phagosomen, aber auch im Zytosol detektierbar sind. Verknüpft mit CPPs werden große Moleküle mit hoher Effizienz von Zellen aufgenommen, die die der meisten konventionellen Transfektionsmethoden übertrifft. In dieser Arbeit wurden peptidgekuppelte siRNAs (pepsiRNAs) als neue Methode zur transienten RNAi in Säugerzellen entwickelt. Hierzu wurden die Peptide über eine Disulfidbrücke mit den siRNAs verknüpft, unter den reduzierenden Bedingungen des Zytosols gespalten wird und , so dass die siRNA im Innern der Zelle freigesetzt wird. PepsiRNAs werden von einer Reihe von Zelltypen aufgenommen, die mit konventionellen Techniken nur schlecht behandelbar sind. Eine deutliche Herunterregulation der Ziel-Genprodukte wurde in einem Konzentrationsbereich von 10 -100 nM beobachtet. SiRNAs mit einer 5´-Thiolmodifikation am sense-Strang wurden durch in vitro Transkription gewonnen, für die ein thiolmodifizierten Nucleotid synthetisiert und dessen Synthese optimiert wurde. Die zellpenetrierenden Peptide Penetratin und Tat wurden rekombinant als Fusionproteine mit Gluatathion-S-Transferase (GST) exprimiert und mit modifizierten Verfahren gereinigt, um die starken Wechselwirkungen der GST-CPPs mit den Zellmembranen zu überwinden. Um die CPPs vom GST-Fusionstag zu trennen, wurde rekombinante TEV-Protease exprimiert und die Aktivität mit der von kommerziell erhältlicher TEV-Protease verglichen. Somit stehen nun Alternativen zur Festphasensynthese zur Verfügung, um die Bestandteile der pepsiRNAs in größeren Mengen herzustellen. Schließlich wurden kleine Moleküle mit zellpenetrierenden Eigenschaften als Ersatz für die Peptideinheit entwickelt. Es konnte gezeigt werden, dass fluoreszenzmarkierte Peptoide verschiedener Länge mit aminofunktionalisierten Seitenketten in Konzentrationen von einigen 10 µM von verschiedenen Säugerzelllinien durch einen endozytoseabhängigen Mechanismus aufgenommen werden. Selbst kleine positiv geladene Moleküle wie fluoresceinmarkiertes Spermin werden über einen endozytoseartigen Mechanismus aufgenommen. Auf diese Weise konnten auch Porphyrine ins Innere der Zellen gebracht werden, wo sie bei Belichtung ihre zytotoxische Wirkung entfalten, so dass sie als neuartiger Wirkstoff für die Photodynamische Therapie weiterentwickelt werden können

Chemical synthesis and enzymatic incorporation of artificial nucleotides

Deutsche Übersetzung des Titels: Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotide

Incorporation of 2'-amido-nucleosides in oligodeoxynucleotides and oligoribonucleotides as a model for 2'-linked conjugates

The functionalisation of oligodeoxynucleotides and oligoribonucleotides by incorporation of 2'-amido-2'-deoxyribonucleosides, possibly containing a reporter group via the 2'-amido bond, was examined. Therefore 2'-acetamido-ribonucleosides containing a small methyl group at the 2'-amido bond were synthesized as model compounds. In order to evaluate the influence of this 2'-modification on the hybridization capacities, 2'-acetamido-2'-deoxyuridine was incorporated in both RNA and DNA strands. The suitability of phosphoramidite chemistry for the introduction of this modified nucleoside was proven using laser desorption mass spectrometry of the final oligonucleotide. The presence of the 2'-modification destabilised both RNA-RNA, DNA-DNA and mixed duplexes. Therefore, it can be concluded that the 2'-acetamido group is not a good linker for attachment of reporter groups to oligonucleotides.status: publishe

Incorporation of 2'-amido-nucleosides in oligodeoxynucleotides and oligoribonucleotides as a model for 2'-linked conjugates.

The functionalisation of oligodeoxynucleotides and oligoribonucleotides by incorporation of 2'-amido-2'-deoxyribonucleosides, possibly containing a reporter group via the 2'-amido bond, was examined. Therefore 2'-acetamido-ribonucleosides containing a small methyl group at the 2'-amido bond were synthesized as model compounds. In order to evaluate the influence of this 2'-modification on the hybridization capacities, 2'-acetamido-2'-deoxyuridine was incorporated in both RNA and DNA strands. The suitability of phosphoramidite chemistry for the introduction of this modified nucleoside was proven using laser desorption mass spectrometry of the final oligonucleotide. The presence of the 2'-modification destabilised both RNA-RNA, DNA-DNA and mixed duplexes. Therefore, it can be concluded that the 2'-acetamido group is not a good linker for attachment of reporter groups to oligonucleotides