Ralf Seidel

We evaluate strategies to quantitatively image quasi-1D and 3D DNA origami on thin carbon films using transmission electron microscopy and show that scanning-mode STEM is the optimal approach for high-contrast imaging even of unstained samples.

DNA-based technologies rely on short, transient hybridization events, but selecting sequences with desired kinetic properties remains largely empirical because hybridization kinetics are difficult to predict from sequence and slow to measure one sequence at a time. Here, we introduce SPARXS-Hyb, an implementation of SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space) for multiplexed sequence-resolved screening of DNA hybridization. Using a surface-immobilized docking-strand library and a quencher-labelled imager-strand library, we screened 128 different DNA sequences in a single kinetic measurement, exposing all sequences to identical experimental conditions. This multiplexed approach removes a major confounding factor of serial measurements, allowing sequence-dependent differences to be compared directly. The resulting dataset reveals sequence-dependent transient binding behaviours and enabled us to identify a sequence with which an order-of-magnitude higher sampling rate can be achieved in DNA-PAINT (DNA points accumulation for imaging in nanoscale topography), a super-resolution microscopy technique based on DNA hybridization. By enabling multiplexed screening across a sequence library, SPARXS-Hyb provides a route to kinetics-guided sequence selection for programmable transient interactions in DNA nanotechnology.

Bottom‐up fabrication of inorganic nanostructures is emerging as an alternative to classical top‐down approaches, offering precise nanometer‐scale control at relatively low cost and effort. In particular, DNA nanostructures provide versatile scaffolds for directly templating the growth of metal structures. Previously, a DNA mold‐based method for metal nanostructure synthesis has been established that supports a modular structure design and a high control over the structure formation. So far, this method has been limited to the growth of gold and palladium nanostructures. Here, we report on the successful adaptation of the DNA mold‐based fabrication method to produce continuous silver nanowires. By optimizing reagent concentrations and applying gentle thermal annealing, we obtain continuous wire structures of several hundred nanometer in length and 27 nm in diameter, overcoming limitations in anisotropic growth. Electric measurements reveal, however, an insulating behavior of these wires. Detailed material analysis shows that the structures initially consist of metallic silver. Upon the deposition on a solid substrate, the silver phase becomes contaminated mainly by atmospheric sulfur, accompanied by structural changes of the wire. This work demonstrates the versatility of DNA‐based metallization and, at the same time highlights the importance of optimizing post‐processing and contacting conditions for successful device fabrication using silver nanoelectrodes.

Downscaling nanoelectronic circuits with conventional lithography becomes increasingly demanding. The quest for less laborious and more sustainable alternatives has sparked investigations attempting to self‐assemble electronics. DNA origami molds offer an exceptional platform for templating metallic nanostructures, enabling programmability and spatial control over nanowire formation. For electrical interconnects, a range of materials capable of forming reliable ohmic contacts is essential. Here, we present the self‐assembly and full electrical characterization of Pd nanowires based on DNA origami molds. The nanowires exhibit metallic behavior showing linear current–voltage characteristics and a decreasing resistance with lowering temperature. While annealing strategies lead to a decrease in conductance, structural rearrangements induced by the applied electric field can either enhance or inhibit charge transport. These findings highlight the potential of DNA‐templated Pd nanowires as self‐assembled metallic interconnects with tuneable electric properties suitable for nanoelectronic circuits.

Superparamagnetic beads are used in single‐molecule magnetic tweezers experiments to investigate the mechanics and dynamics of biomolecules. The beads exhibit a weak anisotropy, such that they align with the applied magnetic field. This allows to rotate the beads and thus to twist of attached biomolecules. To quantitatively understand the origin of the magnetization anisotropy, we use high‐speed magnetic tweezers experiments, numerical simulations, as well as fluxgate magnetorelaxometry measurements. We find that Brownian orientation fluctuations of the beads occur up to cut‐off frequencies of ∼100 Hz being only weakly dependent on the applied field, which superimpose the dynamics of attached biomolecules. When simulating the equilibrium magnetization of single beads as ensembles of ∼10 ⁵ anisotropic and randomly oriented superparamagnetic nanoparticles, a single anisotropy axis is formed, which matches the magnitude of the experimental results. The time scale of the magnetization relaxation spans several orders of magnitude. Overall, our data reveal that the magnetic beads contain randomly oriented magnetic domains with a rather wide size distribution in which the bead anisotropy is the finite residual of the sum of the nanoparticle anisotropies. It is thus an inherent property of magnetic beads and needs to be considered in high‐resolution measurements of magnetic tweezers.

Bottom-up fabrication of inorganic nanostructures is emerging as an alternative to classical top-down approaches, offering precise nanometer-scale control at relatively low cost and effort. In particular, DNA nanostructures provide versatile scaffolds for directly templating the growth of metal structures. Previously, a DNA mold-based method for metal nanostructure synthesis has been established that supports a modular structure design and a high control over the structure formation. So far, this method was limited to the growth of gold and palladium nanostructures. Here, we report the successful adaptation of the DNA mold-based fabrication method to produce continuous silver nanowires. By optimizing reagent concentrations and applying gentle thermal annealing, we obtain continuous wire structures of several hundred nanometer length, overcoming limitations in anisotropic growth. Despite the strong interaction of silver ions with DNA, we can control the growth without increasing the complexity of our approach. Our structures are not oxidized yet they did not exhibit conductivity. This work demonstrates the versatility of DNA-templated metallization and opens new opportunities for constructing self-assembled hybrid nanostructures with controlled shape and composition.

Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 functions as a homodimer bound to a single ∼200 nt RNA. While the basic biochemical properties of Cas12f1, such as its use of a single catalytic center for catalysis, have been characterized, the orchestration of the different events occurring during Cas12f1 reactions remained little explored. To gain insights into the dynamics and mechanisms involved in DNA recognition and cleavage by Cas12f1 from Syntrophomonas palmitatica (SpCas12f1), we solved the structure of SpCas12f1 bound to target DNA and employed single-molecule magnetic tweezers measurements in combination with ensemble kinetic measurements. Our data indicate that SpCas12f1 forms 18 bp R-loops, in which local contacts of the protein to the R-loop stabilize R-loop intermediates. DNA cleavage is catalyzed by a single SpCas12f1 catalytic center, which first rapidly degrades a ∼11 bp region on the nontarget strand by cutting at random sites. Subsequent target strand cleavage is slower and requires at least a nick in the nontarget strand.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5′-terminated strands at DSB sites^1,2. The breast cancer suppressor BRCA1–BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress^{1,3,4,5,6,7,8,9}. BRCA1–BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear^{10,11,12,13,14,15,16}. Using purified recombinant proteins, we show here that BRCA1–BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1–BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11–RAD50–NBS1 and phosphorylated CtIP, BRCA1–BARD1 forms the BRCA1–C complex^17,18, which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1–C complex^19,20,21, inhibits resection, showing that BRCA1–C is a functionally integrated ensemble. Whereas BRCA1–BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. ^4,5,6,8). We show that in the presence of RAD51, BRCA1–BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1–BARD1 in various physiological contexts.

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.