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Biomedical subjects

Christof M Niemeyer

Publications and source records attributed to Christof M Niemeyer.

At least 19 recordsLinked to original sources
PubMed2007

Microtiter plate-based screening for the optimization of DNA-protein conjugate synthesis by means of expressed protein ligation.

We report a rapid microtiter plate screening assay for the optimization of the synthesis of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter containing a C-terminal thioester that enables a mild and highly specific reaction with N-terminal cysteine compounds. To screen for optimal reaction conditions, we developed a microtiter plate-based assay that utilizes DNA-directed immobilization of the products formed in the ligation reaction of cysteine-modified DNA oligonucleotides with the model protein thioester of the maltose-binding protein (MBP), recombinantly expressed as an intein-fusion protein in E. coli. The screening assay allowed the rapid quantitative monitoring of various reaction parameters, such as the ratio of the reactants, reaction times, pH and ion strength of the buffer, the influence of various thiol additives and the nature of the chemical linker within the cysteine-bearing DNA oligonucleotide. As the consequence of the assay-based optimization, the ligation of MBP with the oligonucleotide was improved to near quantitative yields.

Biochemistry↗
PubMed2006

Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor.

The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.

Animals↗
PubMed2006

A generic building block for C- and N-terminal protein-labeling and protein-immobilization.

Expressed protein ligation (EPL) and bioconjugation based on the maleimide group (MIC-conjugation) provide powerful tools for protein modification. In the light of the importance of site-selectively modified proteins for the study of protein function, a flexible method for the introduction of tags and reporter groups into the C-terminus of proteins employing EPL and MIC-conjugation was developed. We describe the solid-phase synthesis of a generic building block, equipped with fluorescence markers or different functional groups. This generic building block allows for a flexible incorporation of different tags into proteins and was used for the introduction of fluorescence markers into the C-terminus of Rab and Ras GTPases by EPL or MIC-conjugation techniques. In addition, a building block appropriately modified for the incorporation of an azide into proteins was synthesized. Azide-functionalized Ras protein was immobilized on a phosphane-modified surface by means of Staudinger ligation providing a highly chemoselective ligation method for the immobilization of proteins.

Azides↗
PubMed2006

Dynamic light-scattering analysis of the electrostatic interaction of hexahistidine-tagged cytochrome P450 enzyme with semiconductor quantum dots.

Currently, there is great interest in the development of methods suitable for determining the stoichiometry of biomolecules attached to nanoparticles. We describe the use of the dynamic light-scattering technique (DLS) to determine the stoichiometry of the protein cytochrome P450(BSbeta) attached to CdS and CdSe quantum dots (QDs). The enzyme-conjugated QDs have different diffusion characteristics compared to the QD and enzyme precursors, expressed in their size, scattering intensity as well as zeta-potential values. The significant enhancement of the scattering intensity of QDs observed upon conjugation with the P450(BSbeta) due to the refractive-index increment and the systematic variation in zeta potential resulting from charge neutralization of the anionic QDs by the cationic histidine-tagged P450(BSbeta) have been used for stoichiometry determination.

Anions↗
PubMed2006

Rational design of DNA nanoarchitectures.

DNA has many physical and chemical properties that make it a powerful material for molecular constructions at the nanometer length scale. In particular, its ability to form duplexes and other secondary structures through predictable nucleotide-sequence-directed hybridization allows for the design of programmable structural motifs which can self-assemble to form large supramolecular arrays, scaffolds, and even mechanical and logical nanodevices. Despite the large variety of structural motifs used as building blocks in the programmed assembly of supramolecular DNA nanoarchitectures, the various modules share underlying principles in terms of the design of their hierarchical configuration and the implemented nucleotide sequences. This Review is intended to provide an overview of this fascinating and rapidly growing field of research from the structural design point of view.

DNA, Single-Stranded↗
PubMed2006

A single-molecule Förster resonance energy transfer analysis of fluorescent DNA-protein conjugates for nanobiotechnology.

The development of nanobiotechnological devices requires the ability to build various components with nanometer accuracy. DNA is a well-established nanoscale building block that self assembles due to specific interactions that are encoded in its sequence. Recently, it has become possible to couple proteins to DNA, thereby expanding the capabilities of DNA for use with molecular photonics and bioelectronics. Here, we present the design and characterization of a supramolecular Förster resonance energy transfer (FRET) system by using a fluorescent protein bound to single-stranded DNA (ssDNA), a fluorophore attached to a second ssDNA molecule, and a complementary strand for hybridizing the two fluorophores together. The FRET efficiency was studied by using both ensemble and single-pair FRET measurements. The distance between the two fluorophores was determined from the single-pair FRET efficiency and could be described by a simple cylindrical model for the DNA. Hence, DNA can be used as a scaffold for positioning fluorescent proteins, as well as traditional fluorophores, with nanometer accuracy and shows great potential for use in the future of nanobiotechnology.

Biotechnology↗
PubMed2006

Design and evaluation of single-stranded DNA carrier molecules for DNA-directed assembly.

Due to the exceptional molecular recognition properties of nucleic acids, the computational design of DNA sequence motifs is of paramount interest for a wide variety of applications, ranging from DNA-based nanotechnology and DNA computing to the broad field of DNA microarray technologies. These applications rely on the specificity of Watson-Crick base-pairing, and thus, are highly sensitive to non-specific interactions and the formation of any undesired secondary structures, which contradict an efficient intermolecular hybridization. Here we report on the in silico design and in vitro evaluation of single-stranded DNA (ssDNA) carrier strands for the directional DNA-based positioning of streptavidin (STV) conjugates covalently tagged with short ssDNA oligonucleotides. Each such carrier strand consists of four hybridization sites complementary to the conjugate DNA strands. The high and homogeneous hybridization efficiency measured in vitro by microarray hybridization assays confirms the quality of our in silico sequence design method. Hybridization efficiency of DNA-STV-conjugates depends on the position of the hybridization site in the carrier sequence, where the positions nearest to and farthest from the microarray surface proved to be most favorable.

Carrier Proteins↗
PubMed2005

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation.

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

Base Sequence↗
PubMed2005

Detection of Rotavirus from stool samples using a standardized immuno-PCR ("Imperacer") method with end-point and real-time detection.

Immuno-PCR (IPCR) has been studied to increase the detection sensitivity of current enzyme-linked immuno-sorbent assays (ELISA) as a novel approach for the early detection of Rotavirus infection, a major source for serious diarrhoea for susceptible risk groups. IPCR utilizes specific antibody-DNA conjugates with subsequent amplification of the marker-DNA. An antibody-DNA conjugate specific for Rotavirus antigen VP6 was synthesized and used in combination with a commercially available Rotavirus-ELISA kit. IPCR was carried out using reagents and protocols of the standardized Imperacer system. Real-time PCR monitoring of the marker-DNA amplification was compared to endpoint quantification of amplified haptene-labeled PCR products, using a microtiterplate-based PCR-ELISA. In spiked calibration samples, as few as 100 virus particles/ml could be clearly detected using the IPCR method and either real-time or end-point quantification compared to about 100,000 virus particles/ml in ELISA. Rotavirus positive and negative stool samples were correctly identified by IPCR with a clear separation even of a 10,000-fold dilution of the positive stool samples from the negative control.

Computer Systems↗