Christof M. Niemeyer

Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science

Based on fundamental chemistry, biotechnology and materials science have developed over the past three decades into todays powerful disciplines which allow the engineering of advanced technical devices and the industrial production of active substances for pharmaceutical and biomedical applications. This review is focused on current approaches emerging at the intersection of materials research, nanosciences, and molecular biotechnology. This novel and highly interdisciplinary field of chemistry is closely associated with both the physical and chemical properties of organic and inorganic nanoparticles, as well as to the various aspects of molecular cloning, recombinant DNA and protein technology, and immunology. Evolutionary optimized biomolecules such as nucleic acids, proteins, and supramolecular complexes of these components, are utilized in the production of nanostructured and mesoscopic architectures from organic and inorganic materials. The highly developed instruments and techniques of todays materials research are used for basic and applied studies of fundamental biological processes.

Chemical Strategies for Generating Protein Biochips

Protein biochips are at the heart of many medical and bioanalytical applications. Increasing interest has been focused on surface activation and subsequent functionalization strategies for immobilizing these biomolecules. Different approaches using covalent and noncovalent chemistry are reviewed; particular emphasis is placed on the chemical specificity of protein attachment and on retention of protein function. Strategies for creating protein patterns (as opposed to protein arrays) are also outlined. An outlook on promising and challenging future directions for protein biochip research and applications is also offered.

Nanobiotechnology: Concepts, Applications and Perspectives

Nanobiotechnology :concepts, applications and perspectives , Nanobiotechnology :concepts, applications and perspectives , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Semisynthetic DNA–Protein Conjugates for Biosensing and Nanofabrication

Conjugation with artificial nucleic acids allows proteins to be modified with a synthetically accessible, robust tag. This attachment is addressable in a highly specific manner by means of molecular recognition events, such as Watson-Crick hybridization. Such DNA-protein conjugates, with their combined properties, have a broad range of applications, such as in high-performance biomedical diagnostic assays, fundamental research on molecular recognition, and the synthesis of DNA nanostructures. This Review surveys current approaches to generate DNA-protein conjugates as well as recent advances in their applications. For example, DNA-protein conjugates have been assembled into model systems for the investigation of catalytic cascade reactions and light-harvesting devices. Such hybrid conjugates are also used for the biofunctionalization of planar surfaces for micro- and nanoarrays, and for decorating inorganic nanoparticles to enable applications in sensing, materials science, and catalysis.

Nanopartikel, Proteine und Nucleinsäuren: Die Biotechnologie begegnet den Materialwissenschaften

Auf der Basis der klassischen Chemie haben sich die Materialwissenschaften und die Biotechnologie im Laufe der letzten drei Jahrzehnte zu leistungsfahigen Disziplinen entwickelt, die heutzutage hochkomplexe technische Elemente und Wirkstoffe fur pharmazeutische und biomedizinische Anwendungen im industriellen Masstab produzieren. Dieser Aufsatz beleuchtet aktuelle Forschungsaktivitaten im Grenzgebiet zwischen der Materialforschung, den Nanowissenschaften und der molekularen Biotechnologie. Dieses neue interdisziplinare Gebiet der Chemie ist einerseits eng verknupft mit den physikalischen und chemischen Eigenschaften von organischen und anorganischen Nanopartikeln und betrifft andererseits die verschiedenen Aspekte molekularer Klonierung, rekombinanter DNA- und Proteintechnologie und der Immunologie. Evolutionar optimierte Biomolekule, z. B. Nucleinsauren, Proteine und supramolekulare Komplexe aus diesen Komponenten, werden fur den Aufbau nanostrukturierter und mesoskopischer Architekturen aus organischen und anorganischen Materialien genutzt. Die hochentwickelten Instrumente und Verfahren der modernen Materialforschung konnen eingesetzt werden, um fundamentale biologische Prozesse zu untersuchen.

Covalent DNA–Streptavidin Conjugates as Building Blocks for Novel Biometallic Nanostructures

Supramolecular aggregates of DNA, RNA, streptavidin, immunoglobulin, and nanocrystalline metal clusters can be generated by self-assembly on the basis of oligonucleotide hybridization (shown schematically). Following selective immunosorption on surface-immobilized antigen, the biometallic hybrid is detectable by electron microscopy.

Dendrimer-activated solid supports for nucleic acid and protein microarrays.

The generation of chemically activated glass surfaces is of increasing interest for the production of microarrays containing DNA, proteins, and low‐molecular‐weight components. We here report on a novel surface chemistry for highly efficient activation of glass slides. Our method is based on the initial modification of glass with primary amino groups using a protocol, specifically optimized for high aminosilylation yields, and in particular, for homogeneous surface coverages. In a following step the surface amino groups are activated with a homobifunctional linker, such as disuccinimidylglutarate (DSG) or 1,4‐phenylenediisothiocyanate (PDITC), and then allowed to react with a starburst dendrimer that contains 64 primary amino groups in its outer sphere. Subsequently, the dendritic monomers are activated and crosslinked with a homobifunctional spacer, either DSG or PDITC. This leads to the formation of a thin, chemically reactive polymer film, covalently affixed to the glass substrate, which can directly be used for the covalent attachment of amino‐modified components, such as oligonucleotides. The resulting DNA microarrays were studied by means of nucleic acid hybridization experiments using fluorophor‐labeled complementary oligonucleotide targets. The results indicate that the novel dendrimer‐activated surfaces display a surface coverage with capture oligomers about twofold greater than that with conventional microarrays containing linear chemical linkers. In addition, the experiments suggest that the hybridization occurs with decreased steric hindrance, likely a consequence of the long, flexible linker chain between the surface and the DNA oligomer. The surfaces were found to be resistant against repeated alkaline regeneration procedures, which is likely a consequence of the crosslinked polymeric structure of the dendrimer film. The high stability allows multiple hybridization experiments without significant loss of signal intensity. The versatility of the dendrimer surfaces is also demonstrated by the covalent immobilization of streptavidin as a model protein.

Photochemical Surface Patterning by the Thiol-Ene Reaction†

The immobilization of proteins on solid substrates while controlling the size and dimensions of the generated patterns is increasingly relevant in biotechnology. Site-specific immobilization and thus control over the orientation of proteins is particularly important because, as opposed to nonspecific adsorption, it generates homogeneous surface coverage and accessibility to the active site of the protein. Consequently, different types of bioorthogonal reactions have been developed to attach proteins site-specifically to surfaces and to control protein patterning. Herein, we report the photochemical coupling of olefins to thiols to generate a stable thioether bond for the covalent surface patterning of proteins and small molecules. This reaction has been applied previously in solution for carbohydrate and peptide coupling. The thiol-ene photoreaction proceeds at close to visible wavelengths (l = 365–405 nm) and in buffered aqueous solutions. As a result of its specificity for olefins, this photoreaction can be considered to be bioorthogonal, unlike other photochemical methods used previously for protein immobilization. To adopt the thiol-ene reaction for the immobilization of biomolecules, surfaces functionalized with thiols and biomolecules derivatized with olefins were prepared (Figure 1). Polyamidoamine (PAMAM) dendrimers were attached covalently to silicon oxide surfaces. An aminocaproic acid spacer was attached to the dendrimers to create distance from the surface. Cystamine was coupled to the spacer, and subsequent reduction of the disulfide yielded the desired thiolterminated surfaces. A liquid layer of terminal-olefinfunctionalized molecules dissolved in ethylene glycol was spread onto these wafers, which were then covered immediately with a photomask. Subsequent irradiation of the surfaces through the photomask led to patterning with adducts of covalently attached thioethers. To establish the method, we photochemically attached the biotin derivative 1 to a thiol-functionalized surface as described above (Figure 1). After the removal of unreacted biotin molecules, the surface was incubated with Cy5-labeled streptavidin (SAv) to produce a SAv-patterned surface. Fluorescence images of the resulting surface (Figure 1) demonstrated that lateral gradients and patterns with micrometer-sized features (5–100 mm) over areas of centimeters in width (Figure 1A) were readily accessible. Figure 1B,C and the fluorescence-intensity profile in Figure 1D show that the patterns have a well-defined shape and are homogeneous over large distances. When prolonged sonication (4 h) and stringent washing were carried our after irradiation, SAv patterns with similar fluorescence intensities were observed, whereas control experiments with biotin that lacked the olefin linker showed no distinctive SAv patterns. These results indicate that the covalent attachment of biotin to the surface occurs specifically through the proposed thiol-ene reaction and that the nonspecific adsorption of biotin is insignificant. Figure 1E shows that the amount of material immobilized can be modified by changing the irradiation time. The procedure reproducibly requires a short irradiation time of 60 s to yield sufficient surface coverage for fabricating dense SAv patterns. To obtain homogeneous fluorescence signals of the patterns, the starting concentration of the solution that is drop cast onto the surface is also important. When the solution of 1 was diluted (to 1 mm), the Cy5-fluorescence intensity decreased considerably. Further dilution (below 500 mm) resulted eventually in disrupted SAv patterns. The application of more concentrated solutions of 1 (> 20 mm) resulted in the saturation of the fluorescence intensity of the SAv patterns. This behavior corresponds well with the effects observed upon varying the irradiation time. Longer irradi[*] Dr. D. N sse, Dr. H. Schroeder, Dr. R. Wacker, Prof. Dr. C. M. Niemeyer Faculty of Chemistry Biological-Chemical Microstructuring Technical University of Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax: (+49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de

Orthogonal Protein Decoration of DNA Origami

Structural DNA nanotechnology 2] and the technique of DNA origami enable the rapid generation of a plethora of complex self-assembled nanostructures. Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years. The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibody– antigen interactions, 9] aptamer binding, 11] nucleic acid hybridization of DNA-tagged proteins, 13] or predominantly biotin–streptavidin (STV) interactions. We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin–STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O-alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as “Snap-tag”, or haloalkane dehalogenase, which is also known as “HaloTag”. By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins. To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, and the commercially available HaloTag system. The respective smallmolecule suicide tags (O-benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1b). To gain access to fusion proteins bearing the complementary Snapand Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) and mKate, the enzymes cytochrome C peroxidase (CCP) and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein “covalin”, a chimera which specifically reacts with both BG and CH, as well as monovalent STV (mSTV), were used in this study. The fusion proteins were overexpressed and purified by conventional procedures (see the Supporting Information). The coupling of BGand CHmodified oligonucleotides to the protein was analyzed by using covalin as the initial model to simplify the electrophoretic characterization. It is shown in Figure 1c that both BGand CH-modified single-stranded DNA (ssDNA) oligonucleotides couple effectively to generate the corresponding DNA–covalin conjugates in nearly quantitative yields. DNA coupling of the aforementioned POI fusions, namely mKateSnap, EST2-SNAP, mKate-Halo, CCP-Halo, and EYFP-Halo occurred in a highly specific manner (Figure 1d), and neither Snap or Halo nor mSTV revealed cross-reactivity for the orthogonal-tagged DNA oligomers. We then used SARSE software to aid in the design of face-shaped DNA origami to demonstrate the selective immobilization of protein on DNA nanostructures. Correct folding of M13mp18 ssDNA through the use of 236 staple strands was analyzed by atomic force microscopy (AFM; details of the sequence design as well as experimental procedures are reported in the Supporting Information). Figure 2a illustrates that the face-shaped DNA origami was obtained in high purity, and high-resolution AFM clearly revealed the proposed ears, neck, and seam features of this structure. As an initial test for protein decoration, we selected 23 staple strands, which were biotinylated to create eyes (2 6 [*] Dr. B. Sacc , Dipl.-Chem. R. Meyer, Dipl.-Biotechnol. M. Erkelenz, M. Sc. K. Kiko, A. Arndt, Dr. H. Schroeder, Dr. K. S. Rabe, Prof. C. M. Niemeyer Technische Universit t Dortmund, Fakult t Chemie Biologisch-Chemische Mikrostrukturtechnik Otto-Hahn Strasse 6, 44227 Dortmund (Germany) Fax: (+ 49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de [] These authors contributed equally to this work.

DNA-Directed Assembly of Bienzymic Complexes from In Vivo Biotinylated NAD(P)H:FMN Oxidoreductase and Luciferase

The DNA-directed assembly of proteins offers a promising route to the generation of spatially ordered multienzyme complexes (MECs), which are not accessible by conventional chemical crosslinking or genetic engineering. MECs with several catalytic centers arranged in a spatially defined way are abundant in nature. Mechanistic advantages of MECs are revealed during the multistep catalytic transformation of a substrate since reactions limited by the rate of diffusional transport are accelerated by the immediate proximity of the catalytic centers. Furthermore, the TMsubstrate-channeling∫ of intermediate products avoids side reactions. Artifical multienzymes would allow the development of novel catalytic systems for enzyme process technology that are capable of regenerating cofactors, as well as multistep chemical transformations; they are also useful for exploration of proximity effects in biochemical pathways. Herein we report the initial steps towards the development of artificial multienzyme complexes through the DNA-directed assembly of two enzymes, NAD(P)H:FMN Oxidoreductase (NFOR) and Luciferase (Luc), which catalyze two consecutive reaction steps (Figure 1). NFOR reduces flavin mononucleotide Figure 1. Schematic representation of the bienzymic reaction cascade catalyzed by NAD(P)H:FMN Oxidoreductase (NFOR) and Luciferase (Luc). R CH3(CH2)10 .