Selective targeting and degradation of doxorubicin-loaded folate-functionalized DNA nanocages.

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.

Fluorescent labelling of in situ hybridisation probes through the copper-catalysed azide-alkyne cycloaddition reaction.

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.

One-Step Formation of "Chain-Armor"-Stabilized DNA Nanostructures.

DNA-based self-assembled nanostructures are widely used to position organic and inorganic objects with nanoscale precision. A particular promising application of DNA structures is their usage as programmable carrier systems for targeted drug delivery. To provide DNA-based templates that are robust against degradation at elevated temperatures, low ion concentrations, adverse pH conditions, and DNases, we built 6-helix DNA tile tubes consisting of 24 oligonucleotides carrying alkyne groups on their 3'-ends and azides on their 5'-ends. By a mild click reaction, the two ends of selected oligonucleotides were covalently connected to form rings and interlocked DNA single strands, so-called DNA catenanes. Strikingly, the structures stayed topologically intact in pure water and even after precipitation from EtOH. The structures even withstood a temperature of 95 °C when all of the 24 strands were chemically interlocked.

Oligothiophenes as fluorescent markers for biological applications.

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

A new ground state single electron donor for excess electron transfer studies in DNA.

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.

Cellular uptake of covalent and non-covalent DNA nanostructures with different sizes and geometries.

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.

Postsynthetic DNA modification through the copper-catalyzed azide-alkyne cycloaddition reaction.

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.

Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures.

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.

Cellular Uptake of Tile-Assembled DNA Nanotubes.

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

Independent generation of C5'-nucleosidyl radicals in thymidine and 2'-deoxyguanosine.

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. 1x10(6) s-1 and is highly stereoselective affording only the (5'S)-diastereomer.

Preparation of bicyclo[3.2.0]heptane-2-endo,7-endo-diols: 1,3-diols with a chiral rigid backbone.

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.