Günter von Kiedrowski

DNA nanotechnology: Chemical copying of connectivity

Three-dimensional DNA nanoscaffolds such as supramolecular tetrahedra can self-assemble from tris-oligonucleotidyls — synthetic three-armed building blocks in which three identical or non-identical short DNA sequences are connected by a tris-linking backbone. Here we show that the connectivity information contained in these building blocks can be copied by using template-directed tris-linking. This finding is a crucial step towards the replication of nanoarchitectures that are based on tris-oligonucleotidyls and to the realization of artificially self-replicating systems on a nanometre scale.

Self-Assembly of a DNA Dodecahedron from 20 Trisoligonucleotides with C3h Linkers†

Since Seeman s pioneering work, DNA has been recognized as a building material for programmable hollow 3D nanoobjects. DNA nanoconstruction benefits from the structural rigidity of short DNA double strands, scalability, good accessibility of synthetic and chemically modified DNA, and the option for enzymatic amplification and processing. Four strategies for the construction of nanoobjects such as polyhedra exist so far. Strategy I is vertex-centered and goes from noncovalent junctions to covalent objects: Noncovalent three-way junctions are assembled from three linear oligonucleotides. Each arm contains a sticky end sequence which is hybridized to its complement in another junction and then covalently connected by DNA ligases. Strategy II is the reverse of strategy I and makes use of trisoligonucleotides, in other words covalent junctions are noncovalently assembled to give the target nanoobjects. Non-natural modes of copying and amplifying were proposed to enable the replication of junctions and nanoconstructs from the latter. Strategy III is a face-centered approach, employing as many oligonucleotides as there are faces on the object while each oligonucleotide is composed of as many segments as there are edges surrounding the faces. Strategy IV first defines the longest path through the object by connecting all vertices using a very long DNA single strand; a set of shorter oligonucleotides generates suitable rigid motifs such as double crossovers, while additional connectivities are expressed by means of paranemic crossover motifs. Recently the assembly of triangular prisms, cubes, pentameric and hexameric prisms, heteroprisms, and biprisms was reported. A set of single-stranded linear and cyclic DNA building blocks was used, and in the latter case rigid organic linker molecules were used as vertices. Herein we report on a new generation of trisoligonucleotides and their employment in benchmark experiments to evaluate strategy II. We selected a dodecahedron, as polyhedra with a smaller number of vertices have been described already. The feasibility of constructing a dodecahedron that reflects the basic symmetry of a virus was forseen for strategy III, but so far this has not been achieved by any strategy. Previously prepared trisoligonucleotides with three different arms were based on asymmetric linker constructs, f,6] so that in principle a set of three different sequences could be connected in three different ways. Linker scaffolds with C3h symmetry are thought to be advantageous because all vertices are expected to be subject to the same conformational constraints. Moreover, diastereomeric mixtures obtained by the utilization of commercially available racemic linker amidites are avoided here. Until now trisoligonucleotides with C3h linkers were synthesized by chemical copying of connectivity, that is the usage of a 3’-connected trisoligonucleotide template for the trislinking of suitable 5’-functionalized linear oligonucleotides. C3h-symmetric linker molecules have also been used to synthesize branched oligonucleotides having mixed sequence directions or equal sequence direction but two or three identical sequences. Very recently, a synthesis of branched oligonucleotides with a C3h-symmetric linker has been described, which is similar to our work presented here. Scheme 1 shows the principle of our approach. Target trisoligonucleotides with three different sequences are assembled on a DNA synthesizer by employing a trislinker amidite orthogonally protected with 4,4’-dimethoxytrityl (DMT) and

Biophysical and lipofection studies of DOTAP analogs.

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.

A Stochastic Model of Nonenzymatic Nucleic Acid Replication: “Elongators” Sequester Replicators

The origin of nucleic acid template replication is a major unsolved problem in science. A novel stochastic model of nucleic acid chemistry was developed to allow rapid prototyping of chemical experiments designed to discover sufficient conditions for template replication. Experiments using the model brought to attention a robust property of nucleic acid template populations, the tendency for elongation to outcompete replication. Externally imposed denaturation-renaturation cycles did not reverse this tendency. For example, it has been proposed that fast tidal cycling could establish a TCR (tidal chain reaction) analogous to a PCR (polymerase chain reaction) acting on nucleic acid polymers, allowing their self-replication. However, elongating side-reactions that would have been prevented by the polymerase in the PCR still occurred in the simulation of the TCR. The same finding was found with temperature and monomer cycles. We propose that if cycling reactors are to allow template replication, oligonucleotide phenotypes that are capable of favorably altering the flux ratio between replication and elongation, for example, by facilitating sequence-specific cleavage within templates, are necessary; accordingly the minimal replicase ribozyme may have possessed restriction functionality.

Selbstanordnung von Trisoligonucleotidylen: „Nano-Acetylen” und „Nano-Cyclobutadien”

Rasches AbkuhlenistdasRezept fur die Selbstanordnung von Nanostrukturen aus Trisoligonucleotidylen, einer neuartigen Klasse verzweigter Oligonucleotide, deren 3′-Enden uber einen trifunktionellen Linker verbunden sind. Die Topologie des kleinsten Komplexes ist formal gesehen aquivalent zur Topologie von Acetylen, wenn ein DNA-Doppelstrang als C-C-Bindung angesehen wird. Abgebildet ist ein Modell von Nano-Acetylen.

Combinatorial synthesis of new cationic lipids and high-throughput screening of their transfection properties.

Here we describe the first synthesis–screening approach for the identification and optimization of new cationic lipids for gene transfer in various cell lines. Combinatorial solid‐phase chemistry was used to synthesize a library of new cationic lipids based on 3‐methylamino‐1,2‐dihydroxypropane as the polar, cationic lipid part. As the nonpolar lipid part, different hydrocarbon chains were bound to the amino group of the scaffold and the amino group was further methylated to afford constantly cationic lipids. Lipids were synthesized in both configurations and as racemates, and the counter ions were also varied. By using a fully automated transfection screening method and COS‐7 cells, the cationic lipid N,N‐ditetradecyl‐N‐methyl‐amino‐2,3‐propanediol (KL‐1‐14) was identified as a candidate lipid for the development of an improved transfection reagent. Screening the transfection properties of KL‐1‐14 in numerous combinations with the helper lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) revealed that Chol is the most suitable helper lipid and the best KL‐1‐14/Chol ratio is 0.5–0.7. Compared to the standard transfection lipid N‐[1‐(2,3‐dioleoyloxy)propyl]‐N,N,N‐trimethylammonium methyl sulfate (DOTAP), transfection efficiency was improved by a factor of about 40. Furthermore, by using R‐ and S‐configured KL‐1‐14, it could be shown that the configuration of the lipids had no significant influence on its transfection efficiency. The highest transfection efficiencies were achieved with chloride as the counter ion. The new lipofection reagent was further tested to transfect the cell lines MDA‐MB‐468, MCF‐7, MDCK‐C7, and primary dentritic cells (DC), which are important for the development of new anticancer gene therapy strategies. Even in these cells, KL‐1‐14/Chol (1:0.6) had improved transfection efficiencies, which were about two to four times higher than for DOTAP.

Synthesis of novel phosphoramidite derivatives bearing pyrenyl and dansyl groups

Abstract The synthesis of novel phosphoramidites is reported starting with the readily available 4-(1-pyrenyl)-butanol or 5-(dimethylamino)naphthalene-1-sulfonic acid chloride, respectively. Both amidites can be used on the DNA synthesizer to prepare 5′ - or internally labeled oligonucleotides without an acyl amide linkage to the fluorescent dye.

A Novel Versatile Phosphoramidite Building Block for the Synthesis of 5′- and 3′-Hydrazide Modified Oligonucleotides

We introduce a novel versatile phosphoramidite building block for the modification of oligonucleotides (ONs) with acyl hydrazides on the 5′- or 3′-terminus, or both. The reaction of these hydrazide functionalized ONs with 4-methoxyphenylaldehyde is demonstrated for solution derivatization. Hydrazides are considered nowadays as promising reactants, which show enhanced reactivity at neutral and slightly acidic conditions and higher stability of yielding products as compared to the aliphatic amines, which are broadly used for ONs derivatization. Our method to introduce hydrazides into ONs employs a phosphoramidite modifier designed to split, during ammonia or lithium hydroxide treatment, into two hydrazides via β-elimination of a central bis-2-carbonylethoxysulfone unit. It allows the creation of ONs derivatized with a hydrazide moiety at the 5′-, 3′- and both 5′- and 3′-termini, as well as two different hydrazide containing ONs at the same time, viz. in one sequence on the same solid support. In latter case one can, for example, synthesize two hydrazide containing ONs, where one is 5′-modified and second one is 3′-modified. This work was supported by German Research Council (DFG) and the Federal Ministry of Education and Research (BMBF, Germany). S.I. Antsypovich gratefully acknowledges a stipend and his fellowship from German Academic Exchange Service (DAAD) and Alexander von Humboldt Foundation (AvH-Stiftung, Germany), respectively.

Synthesis of novel mercapto analogues of Tröger's base

Synthesis of 2,8-bis(mercaptomethyl)-6H,12H-5,11-methanodibenzo[bf][1,5]diazocine (2) and its derivatization to 7 and 8 have been reported.

Phase-transfer synthesis of novel water-soluble gold clusters with tripodal thioether based ligands

Tripodal, water-soluble thioethers based on 1,3,5-trismethylbenzene scaffolds are suitable ligands for the Au55 cluster.