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Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.

DNA nanostructures with different sizes and shapes, assembled through either covalent or non-covalent bonds, namely tetrahedral and octahedral nanocages, rod-shaped chainmails, square box and rectangular DNA origami structures, were compared for their stability in serum, cell surface binding, internalization efficiency, and intracellular degradation rate. For cell internalization a specific cell system, highly expressing the scavenger receptor LOX-1 was used. The results indicate that LOX-1 binds and internalizes a broad family of DNA structures of different sizes that, however, have a different fate and lifetime inside the cells. Covalently linked tetrahedra, octahedra or chainmails are intact inside cells for up to 18 hours whilst the same DNA nanostructures without covalent bonds along with square box and rectangular origami are rapidly degraded. These data suggest that non-covalently linked structures may be useful for fast drug release whilst the covalently-linked structures could be appropriate vehicles for slow release of molecules.

In situ hybridisation is a powerful tool to investigate the genome and chromosome architecture. Nick translation (NT) is widely used to label DNA probes for fluorescence in situ hybridisation (FISH). However, NT is limited to the use of long double-stranded DNA and does not allow the labelling of single-stranded and short DNA, e.g. oligonucleotides. An alternative technique is the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), at which azide and alkyne functional groups react in a multistep process catalysed by copper(I) ions to give 1,4-distributed 1,2,3-triazoles at a high yield (also called 'click reaction'). We successfully applied this technique to label short single-stranded DNA probes as well as long PCR-derived double-stranded probes and tested them by FISH on plant chromosomes and nuclei. The hybridisation efficiency of differently labelled probes was compared to those obtained by conventional labelling techniques. We show that copper(I)-catalysed azide-alkyne cycloaddition-labelled probes are reliable tools to detect different types of repetitive sequences on chromosomes opening new promising routes for the detection of single copy gene. Moreover, a combination of FISH using such probes with other techniques, e.g. immunohistochemistry (IHC) and cell proliferation assays using 5-ethynyl-deoxyuridine, is herein shown to be easily feasible.

Sich selbststandig anordnende DNA-basierte Nanostrukturen finden weitverbreitete Anwendung bei der prazisen Positionierung organischer und anorganischer Komponenten im Nanometerbereich. Besonders vielversprechend ist ihre Verwendung als programmierbare Systeme fur den zielgerichteten Wirkstofftransport. Um DNA-basierte Strukturen zu entwickeln, die robust gegen Zersetzung bei erhohten Temperaturen, niedrigen Ionenkonzentrationen, ungunstigen pH-Bedingungen oder durch DNAsen sind, haben wir 6-Helix-Bundel aus 24 Oligonukleotiden konstruiert, die an ihrem 3′-Ende Alkingruppen und an ihrem 5′-Ende Azidgruppen tragen. Mithilfe einer sanften Klick-Reaktion wurden die beiden Enden ausgewahlter Oligonukleotide kovalent miteinander verbunden, um Ringe und verschrankte DNA-Strange zu erzeugen. Auffallenderweise blieben diese DNA-Catenane in Wasser oder selbst nach Ethanolfallung topologisch intakt. Gleiches galt, wenn die Strukturen Temperaturen uber 95 °C ausgesetzt wurden, sofern alle 24 Strange kovalent cyclisiert wurden.

The attachment of labels onto DNA is of utmost importance in many areas of biomedical research and is valuable in the construction of DNA-based functional nanomaterials. The copper(I)-catalyzed Huisgen cycloaddition of azides and alkynes (CuAAC) has recently been added to the repertoire of DNA labeling methods, thus allowing the virtually unlimited functionalization of both small synthetic oligonucleotides and large gene fragments with unprecedented efficiency. The CuAAC reaction yields the labeled polynucleotides in very high purity after a simple precipitation step. The reviewed technology is currently changing the way in which functionalized DNA strands are generated cost-efficiently in high quality for their application in molecular diagnostics systems and nanotechnological research.

Die Markierung von DNA ist von groster Bedeutung fur die Biomedizin und bildet die Basis fur die Konstruktion DNA-basierter funktionaler Nanomaterialien. Seit kurzem zahlt die Kupfer(I)-katalysierte Huisgen-Cycloaddition von Aziden mit Alkinen (CuAAC) zum Repertoire der DNA-Markierungsmethoden. Diese Methode ermoglicht die Modifizierung von DNA mit beispielloser Effizienz. Nicht nur kleine Oligonucleotide, sondern auch ganze Genfragmente konnen so hochgradig chemisch modifiziert werden. Die CuAAC-Reaktion liefert die markierten Polynucleotide, meist nach einfacher Ausfallung, in ausgezeichneter Reinheit und ist auf dem besten Wege, die Synthese funktionalisierter DNA-Strange zu revolutionieren. Die Methode wird von entscheidender Bedeutung fur die Erzeugung neuer diagnostischer Systeme und fur die nanotechnologische Forschung sein.

The use of BrdU (5′-bromo-2′-deoxyuridine) is a widely accepted method for the detection of cell proliferation. Despite the good performance, this method has yet shown some disadvantages such as the destruction of the target DNA in order to allow the antibody-driven colorimetric/fluorescent detection of newly synthesized cells. This harsh DNA destructing attack on the system can be overcome by using EdU (5′-ethynyl-2′-deoxyuridine) as innovative method based on click chemistry.

The easily available bicyclo[3.2.0]hept-3-en-6-ones (1a-f) have been converted into the corresponding bicyclo[3.2.0]heptane-2-endo,7-endo-diols (4a-f) in an efficient and stereoselective fashion. This preparation opens a route to a family of 1,3-diols with a chiral rigid backbone, potentially suitable as nonracemic precursors for bidentate ligands in asymmetric synthesis.

The attachment of labels onto DNA is of utmost importance in many areas of biomedical research and is valuable in the construction of DNA-based functional nanomaterials. The copper(I)-catalyzed Huisgen cycloaddition of azides and alkynes (CuAAC) has recently been added to the repertoire of DNA labeling methods, thus allowing the virtually unlimited functionalization of both small synthetic oligonucleotides and large gene fragments with unprecedented efficiency. The CuAAC reaction yields the labeled polynucleotides in very high purity after a simple precipitation step. The reviewed technology is currently changing the way in which functionalized DNA strands are generated cost-efficiently in high quality for their application in molecular diagnostics systems and nanotechnological research.

The synthesis of the C5' tert-butyl ketone of thymidine 1a and 2'-deoxyguanosine 2 is achieved by reaction of 5'-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5'R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5'S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5'-yl radical and the 2'-deoxyguanosin-5'-yl radical, respectively. In the thymidine system, the C5' radical is fully quenched in the presence of a physiological concentration of thiols. In the 2'-deoxyguanosine system, the C5' radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5',8-cyclo-2'-deoxyguanosine derivative. The cyclization of the 2'-deoxyguanosin-5'-yl radical occurs with a rate constant of ca. 1 x 10(6) s(-1) and is highly stereoselective affording only the (5'S)-diastereomer.

The synthesis of the C5' tert-butyl ketone of thymidine 1a and 2'-deoxyguanosine 2 is achieved by reaction of 5'-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5'R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5'S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5'-yl radical and the 2'-deoxyguanosin-5'-yl radical, respectively. In the thymidine system, the C5' radical is fully quenched in the presence of a physiological concentration of thiols. In the 2'-deoxyguanosine system, the C5' radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5',8-cyclo-2'-deoxyguanosine derivative. The cyclization of the 2'-deoxyguanosin-5'-yl radical occurs with a rate constant of ca. 1 x 10(6) s(-1) and is highly stereoselective affording only the (5'S)-diastereomer.

Efficient methodologies based on consecutive radical reactions for the preparation of cyclonucleosides 5 and 13 are reported. The reactions were performed on modified thymidine and 2′-deoxyadenosine substrates using (TMS)3SiH as the reducing agent. The protected 5′-carbaldehyde 3 afforded the cyclonucleoside 4 in 85 % yield and in a diastereoisomeric ratio 4a/4b = 3:7. The mono-desilylation at the 5′-O position of these cyclonucleosides has been successfully achieved by UV irradiation affording quantitatively derivatives 5a and 5b. The protected 5′-carbaldehyde 10 afforded the cyclonucleoside 12 in 70–75 % yield as a single diastereoisomer (5′R) either in deoxygenated solution, followed by in situ oxidation of the reaction mixture by chloranil, or in aerobic conditions. The photochemical 5′-O-desilylation of 12 has been obtained in 56 % yield affording the cyclopurine 13. The reaction mechanisms have been studied in some detail using a variant of the radical clock methodology. The C5′ radical 6 or 15, generated by addition of the (TMS)3Si· radical to the corresponding aldehyde, undergoes a 6-exo-trig cyclization on the base moiety prior to termination. The rate constants for both 6-exo-trig radical cyclizations have been estimated to be close to 105 s–1 at 86 °C, the cyclization on the purine moiety being 4–5 times faster than that on the pyrimidine group. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

A new photoinducible single electron donor has been developed, which, when linked to thymidine, is shown to be an efficient ground state reducing agent in DNA; the donor can be activated at wavelengths where standard DNA does not absorb.

Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA that is compatible with polymerase enzymes.