Niels V. Voigt

Single-molecule chemical reactions on DNA origami

DNA nanotechnology and particularly DNA origami, in which long, single-stranded DNA molecules are folded into predetermined shapes, can be used to form complex self-assembled nanostructures. Although DNA itself has limited chemical, optical or electronic functionality, DNA nanostructures can serve as templates for building materials with new functional properties. Relatively large nanocomponents such as nanoparticles and biomolecules can also be integrated into DNA nanostructures and imaged. Here, we show that chemical reactions with single molecules can be performed and imaged at a local position on a DNA origami scaffold by atomic force microscopy. The high yields and chemoselectivities of successive cleavage and bond-forming reactions observed in these experiments demonstrate the feasibility of post-assembly chemical modification of DNA nanostructures and their potential use as locally addressable solid supports.

DNA origami: a quantum leap for self-assembly of complex structures

The spatially controlled positioning of functional materials by self-assembly is one of the fundamental visions of nanotechnology. Major steps towards this goal have been achieved using DNA as a programmable building block. This tutorial review will focus on one of the most promising methods: DNA origami. The basic design principles, organization of a variety of functional materials and recent implementation of DNA robotics are discussed together with future challenges and opportunities.

Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

Quantifying Contribution of Synthrophic Acetate Oxidation to Methane Production in Thermophilic Anaerobic Reactors by Membrane Inlet Mass Spectrometry

A unique method was developed and applied for monitoring methanogenesis pathways based on isotope labeled substrates combined with online membrane inlet quadrupole mass spectrometry (MIMS). In our study, a fermentation sample from a full-scale biogas plant fed with pig and cattle manure, maize silage, and deep litter was incubated with 100 mM of [2-(13)C] sodium acetate under thermophilic anaerobic conditions. MIMS was used to measure the isotopic distribution of dissolved CO2 and CH4 during the degradation of acetate, while excluding interference from water by applying a cold trap. After 6 days of incubation, the proportion of methane derived from reduction of CO2 had increased significantly and reached up to 87% of total methane, suggesting that synthrophic acetate oxidation coupled to hydrogenotrophic methanogenesis (SAO-HM) played an important role in the degradation of acetate. This study provided a new approach for online quantification of the relative contribution of methanogenesis pathways to methane production with a time resolution shorter than one minute. The observed contribution of SAO-HM to methane production under the tested conditions challenges the current widely accepted anaerobic digestion model (ADM1), which strongly emphasizes the importance of the acetoclastic methanogenesis.

In-situ biogas upgrading with pulse H2 additions: The relevance of methanogen adaption and inorganic carbon level

Surplus electricity from fluctuating renewable power sources may be converted to CH4 via biomethanisation in anaerobic digesters. The reactor performance and response of methanogen population of mixed-culture reactors was assessed during pulsed H2 injections. Initial H2 uptake rates increased immediately and linearly during consecutive pulse H2 injections for all tested injection rates (0.3 to 1.7LH2/Lsludge/d), while novel high throughput mcrA sequencing revealed an increased abundance of specific hydrogenotrophic methanogens. These findings illustrate the adaptability of the methanogen population to H2 injections and positively affects the implementation of biomethanisation. Acetate accumulated by a 10-fold following injections exceeding a 4:1 H2:CO2 ratio and may act as temporary storage prior to biomethanisation. Daily methane production decreased for headspace CO2 concentrations below 12% and may indicate a high sensitivity of hydrogenotrophic methanogens to CO2 limitation. This may ultimately decide the biogas upgrading potential which can be achieved by biomethanisation.

Micro-scale H2-CO2 Dynamics in a Hydrogenotrophic Methanogenic Membrane Reactor.

Biogas production is a key factor in a sustainable energy supply. It is possible to get biogas with very high methane content if the biogas reactors are supplied with exogenous hydrogen, and one of the technologies for supplying hydrogen is through gas permeable membranes. In this study the activity and stratification of hydrogen consumption above such a membrane was investigated by use of microsensors for hydrogen and pH. A hydrogenotrophic methanogenic community that was able to consume the hydrogen flux within 0.5 mm of the membrane with specific rates of up to 30 m3 H2 m-3 day-1 developed within 3 days in fresh manure and was already established at time zero when analyzing slurry from a biogas plant. The hydrogen consumption was dependent on a simultaneous carbon dioxide supply and was inhibited when carbon dioxide depletion elevated the pH to 9.2. The activity was only partially restored when the carbon dioxide supply was resumed. Bioreactors supplied with hydrogen gas should thus be carefully monitored and either have the hydrogen supply disrupted or be supplemented with carbon dioxide when the pH rises to values about 9.

Single Molecule FRET Analysis of the 11 Discrete Steps of a DNA Actuator

DNA hybridization allows the design and assembly of dynamic DNA-based molecular devices. Such structures usually accomplish their function by the addition of fuel strands that drive the structure from one conformation to a new one or by internal changes in DNA hybridization. We report here on the performance and robustness of one of these devices by the detailed study of a dynamic DNA actuator. The DNA actuator was chosen as a model system, as it is the device with most discrete states to date. It is able to reversibly slide between 11 different states and can in principle function both autonomously and nonautonomously. The 11 states of the actuator were investigated by single molecule Förster Resonance Energy Transfer (smFRET) microscopy to obtain information on the static and dynamic heterogeneities of the device. Our results show that the DNA actuator can be effectively locked in several conformations with the help of well-designed DNA lock strands. However, the device also shows pronounced static and dynamic heterogeneities both in the unlocked and locked modes, and we suggest possible structural models. Our study allows for the direct visualization of the conformational diversity and movement of the dynamic DNA-based device and shows that complex DNA-based devices are inherently heterogeneous. Our results also demonstrate that single molecule techniques are a powerful tool for structural dynamics studies and provide a stringent test for the performance of molecular devices made out of DNA.