Electrokinetic Torque Generation by DNA Nanorobotic Arms Studied via Single-Molecule Fluctuation Analysis.

DNA nanotechnology has enabled the creation of supramolecular machines, whose shape and function are inspired from traditional mechanical engineering as well as from biological examples. As DNA inherently is a highly charged biopolymer, the external application of electric fields provides a versatile, computer-programmable way to control the movement of DNA-based machines. However, the details of the electrohydrodynamic interactions underlying the electrical manipulation of these machines are complex, as the influence of their intrinsic charge, the surrounding cloud of counterions, and the effect of electrokinetic fluid flow have to be taken into account. In this work, we identify the relevant effects involved in this actuation mechanism by determining the electric response of an established DNA-based nanorobotic arm to varying design and operation parameters. Borrowing an approach from single-molecule biophysics, we determined the electrical torque exerted on the nanorobotic arms by analyzing their thermal fluctuations when oriented in an electric field. We analyze the influence of various experimental and design parameters on the "actuatability" of the nanostructures and optimize the generated torque according to these parameters. Our findings give insight into the physical processes involved in the actuation mechanism and provide general guidelines that aid in designing and efficiently operating electrically driven nanorobotic devices made from DNA.

Electrical Actuation of DNA-Based Nanomechanical Systems.

DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.

Energy landscapes of rotary DNA origami devices determined by fluorescence particle tracking.

Biomolecular nanomechanical devices are of great interest as tools for the processing and manipulation of molecules, thereby mimicking the function of nature's enzymes. DNA nanotechnology provides the capability to build molecular analogs of mechanical machine elements such as joints and hinges via sequence-programmable self-assembly, which are otherwise known from traditional mechanical engineering. Relative to their size, these molecular machine elements typically do not reach the same relative precision and reproducibility that we know from their macroscopic counterparts; however, as they are scaled down to molecular sizes, physical effects typically not considered by mechanical engineers such as Brownian motion, intramolecular forces, and the molecular roughness of the devices begin to dominate their behavior. In order to investigate the effect of different design choices on the roughness of the mechanical energy landscapes of DNA nanodevices in greater detail, we here study an exemplary DNA origami-based structure, a modularly designed rotor-stator arrangement, which resembles a rotatable nanorobotic arm. Using fluorescence tracking microscopy, we follow the motion of individual rotors and record their corresponding energy landscapes. We then utilize the modular construction of the device to exchange its constituent parts individually and systematically test the effect of different design variants on the movement patterns. This allows us to identify the design parameters that most strongly affect the shape of the energy landscapes of the systems. Taking into account these insights, we are able to create devices with significantly flatter energy landscapes, which translates to mechanical nanodevices with improved performance and behaviors more closely resembling those of their macroscopic counterparts.

Emergence of Colloidal Patterns in ac Electric Fields.

Suspended microparticles subjected to ac electrical fields collectively organize into band patterns perpendicular to the field direction. The bands further develop into zigzag shaped patterns, in which the particles are observed to circulate. We demonstrate that this phenomenon can be observed quite generically by generating such patterns with a wide range of particles: silica spheres, fatty acid, oil, and coacervate droplets, bacteria, and ground coffee. We show that the phenomenon can be well understood in terms of second order electrokinetic flow, which correctly predicts the hydrodynamic interactions required for the pattern formation process. Brownian particle simulations based on these interactions accurately recapitulate all of the observed pattern formation and symmetry-breaking events, starting from a homogeneous particle suspension. The emergence of the formed patterns can be predicted quantitatively within a parameter-free theory.

Tuning the Diameter, Stability, and Membrane Affinity of Peptide Pores by DNA-Programmed Self-Assembly.

Protein pores recently enabled a breakthrough in bioanalytics by making it possible to sequence individual DNA and RNA strands during their translocation through the lumen of the pore. Despite this success and the overall promise of nanopore-based single-molecule analytics, protein pores have not yet reached their full potential for the analysis and characterization of globular biomolecules such as natively folded proteins. One reason is that the diameters of available protein pores are too small for accommodating the translocation of most folded globular proteins through their lumen. The work presented here provides a step toward overcoming this limitation by programmed self-assembly of α-helical pore-forming peptides with covalently attached single-stranded DNA (ssDNA). Specifically, hybridization of the peptide ceratotoxin A (CtxA) with N-terminally attached ssDNA to a complementary DNA template strand with 4, 8, or 12 hybridization sites made it possible to trigger the assembly of pores with various diameters ranging from approximately 0.5 to 4 nm. Hybridization of additional DNA strands to these assemblies achieved extended functionality in a modular fashion without the need for modifying the amino acid sequence of the peptides. For instance, functionalization of these semisynthetic biological nanopores with DNA-cholesterol anchors increased their affinity to lipid membranes compared to pores formed by native CtxA, while charged transmembrane segments prolonged their open-state lifetime. Assembly of these hybrid DNA-peptides by a template increased their cytotoxic activity and made it possible to kill cancer cells at 20-fold lower total peptide concentrations than nontemplated CtxA.

Complete aggregation pathway of amyloid ß (1-40) and (1-42) resolved on an atomically clean interface.

To visualize amyloid ß (Aß) aggregates requires an uncontaminated and artifact-free interface. This paper demonstrates the interface between graphene and pure water (verified to be atomically clean using tunneling microscopy) as an ideal platform for resolving size, shape, and morphology (measured by atomic force microscopy) of Aß-40 and Aß-42 peptide assemblies from 0.5 to 150 hours at a 5-hour time interval with single-particle resolution. After confirming faster aggregation of Aß-42 in comparison to Aß-40, a stable set of oligomers with a diameter distribution of ~7 to 9 nm was prevalently observed uniquely for Aß-42 even after fibril appearance. The interaction energies between a distinct class of amyloid aggregates (dodecamers) and graphene was then quantified using molecular dynamics simulations. Last, differences in Aß-40 and Aß-42 networks were resolved, wherein only Aß-42 fibrils were aligned through lateral interactions over micrometer-scale lengths, a property that could be exploited in the design of biofunctional materials.

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification.

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2+ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2+ cells in a background of HER2- cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.

A Bio-Inspired Amplification Cascade for the Detection of Rare Cancer Cells.

The main cause of cancer-related death is due to cancer cell spreading and formation of secondary tumors in distant organs, the so-called metastases. Metastatic cancer cells are detectable in the blood of cancer patients as circulating tumor cells (CTC) and may be exploited for prognostic and monitoring purposes, including in breast cancer. Due to their very low frequency, however, their quantitative detection remains a challenge in clinical practice. Nature has developed mechanisms to amplify rare biological events or weak signals, such as intracellular signaling pathways, cytokine networks or the coagulation cascades. At the National Center for Competence in Research (NCCR) in Bio-Inspired Materials we are coupling gold nanoparticle-based strategies with fibrinogen and DNA bio-inspired amplification cascades to develop an in vitro test to specifically and sensitively detect CTCs in patients' blood. In this article, we describe the biological context, the concept of bio-inspired amplification, and the approaches chosen. We also discuss limitations, open questions and further potential biomedical applications of such an approach.

Enhanced Efficiency of an Enzyme Cascade on DNA-Activated Silica Surfaces.

In nature, compartmentalized and spatially organized enzyme cascades are utilized to increase the efficiency of enzymatic reactions. From a technologically relevant perspective, synthetic enzyme systems have to be optimized with emphasis on enzyme activity, productivity, scalability, and ease of use. But the underlying principles and relevant parameters that lead to an enhancement of the activity of enzyme cascades through spatial organization are still under debate. Here, we report on the 10-fold activity enhancement of the GOx-HRP enzyme cascade for the oxidation of luminol, when the enzymes are colocalized on micron-scaled solid scaffolds. Both enzymes were initially assembled and concentrated on DNA origami rectangles and finally further concentrated on the surface of silica particles. We show that each particular component of the designed system contributes to the activity enhancement. Furthermore, we measured an influence of the silica particle length scale on the total productivity by a factor of 5-10, but to a lesser extent on the maximum enzyme activity. Our findings demonstrate that micrometer-sized scaffolds can be used to enhance the efficiency of enzyme-cascades by at least a magnitude and that solid-phase scaffolds enable scalability for technological applications.

Nanopore-Based, Rapid Characterization of Individual Amyloid Particles in Solution: Concepts, Challenges, and Prospects.

Aggregates of misfolded proteins are associated with several devastating neurodegenerative diseases. These so-called amyloids are therefore explored as biomarkers for the diagnosis of dementia and other disorders, as well as for monitoring disease progression and assessment of the efficacy of therapeutic interventions. Quantification and characterization of amyloids as biomarkers is particularly demanding because the same amyloid-forming protein can exist in different states of assembly, ranging from nanometer-sized monomers to micrometer-long fibrils that interchange dynamically both in vivo and in samples from body fluids ex vivo. Soluble oligomeric amyloid aggregates, in particular, are associated with neurotoxic effects, and their molecular organization, size, and shape appear to determine their toxicity. This concept article proposes that the emerging field of nanopore-based analytics on a single molecule and single aggregate level holds the potential to account for the heterogeneity of amyloid samples and to characterize these particles-rapidly, label-free, and in aqueous solution-with regard to their size, shape, and abundance. The article describes the concept of nanopore-based resistive pulse sensing, reviews previous work in amyloid analysis, and discusses limitations and challenges that will need to be overcome to realize the full potential of amyloid characterization on a single-particle level.

Multimodal Assessment of Recurrent MTBI across the Lifespan.

Recurrent mild traumatic brain injuries (mTBI) and its neurological sequelae have been the focus of a large number of studies, indicating cognitive, structural, and functional brain alterations. However, studies often focused on single outcome measures in small cohorts of specific populations only. We conducted a multimodal evaluation of the impact of recurrent mTBI on a broad range of cognitive functions, regional brain volume, white matter integrity, and resting state functional connectivity (RSFC) in young and older adults in the chronic stage (>6 months after the last mTBI). Seventeen young participants with mTBI (age: 24.2 ± 2.8 (mean ± SD)) and 21 group-wise matched healthy controls (age: 25.8 ± 5.4 (mean ± SD)), as well as 17 older participants with mTBI (age: 62.7 ± 7.7 (mean ± SD)) and 16 group-wise matched healthy controls (age: 61.7 ± 5.9 (mean ± SD)) were evaluated. We found significant differences in the verbal fluency between young participants with mTBI and young healthy controls. Furthermore, differences in the regional volume of precuneus and medial orbitofrontal gyrus between participants with mTBI and controls for both age groups were seen. A significant age by group interaction for the right hippocampal volume was noted, indicating an accelerated hippocampal volume loss in older participants with mTBI. Other cognitive parameters, white matter integrity, and RSFC showed no significant differences. We confirmed some of the previously reported detrimental effects of recurrent mTBI, but also demonstrated inconspicuous findings for the majority of parameters.

A self-assembled nanoscale robotic arm controlled by electric fields.

The use of dynamic, self-assembled DNA nanostructures in the context of nanorobotics requires fast and reliable actuation mechanisms. We therefore created a 55-nanometer-by-55-nanometer DNA-based molecular platform with an integrated robotic arm of length 25 nanometers, which can be extended to more than 400 nanometers and actuated with externally applied electrical fields. Precise, computer-controlled switching of the arm between arbitrary positions on the platform can be achieved within milliseconds, as demonstrated with single-pair Förster resonance energy transfer experiments and fluorescence microscopy. The arm can be used for electrically driven transport of molecules or nanoparticles over tens of nanometers, which is useful for the control of photonic and plasmonic processes. Application of piconewton forces by the robot arm is demonstrated in force-induced DNA duplex melting experiments.

Intensive speech and language therapy in patients with chronic aphasia after stroke: a randomised, open-label, blinded-endpoint, controlled trial in a health-care setting.

BACKGROUND: Treatment guidelines for aphasia recommend intensive speech and language therapy for chronic (≥6 months) aphasia after stroke, but large-scale, class 1 randomised controlled trials on treatment effectiveness are scarce. We aimed to examine whether 3 weeks of intensive speech and language therapy under routine clinical conditions improved verbal communication in daily-life situations in people with chronic aphasia after stroke. METHODS: In this multicentre, parallel group, superiority, open-label, blinded-endpoint, randomised controlled trial, patients aged 70 years or younger with aphasia after stroke lasting for 6 months or more were recruited from 19 inpatient or outpatient rehabilitation centres in Germany. An external biostatistician used a computer-generated permuted block randomisation method, stratified by treatment centre, to randomly assign participants to either 3 weeks or more of intensive speech and language therapy (≥10 h per week) or 3 weeks deferral of intensive speech and language therapy. The primary endpoint was between-group difference in the change in verbal communication effectiveness in everyday life scenarios (Amsterdam-Nijmegen Everyday Language Test A-scale) from baseline to immediately after 3 weeks of treatment or treatment deferral. All analyses were done using the modified intention-to-treat population (those who received 1 day or more of intensive treatment or treatment deferral). This study is registered with ClinicalTrials.gov, number NCT01540383. FINDINGS: We randomly assigned 158 patients between April 1, 2012, and May 31, 2014. The modified intention-to-treat population comprised 156 patients (78 per group). Verbal communication was significantly improved from baseline to after intensive speech and language treatment (mean difference 2·61 points [SD 4·94]; 95% CI 1·49 to 3·72), but not from baseline to after treatment deferral (-0·03 points [4·04]; -0·94 to 0·88; between-group difference Cohen's d 0·58; p=0·0004). Eight patients had adverse events during therapy or treatment deferral (one car accident [in the control group], two common cold [one patient per group], three gastrointestinal or cardiac symptoms [all intervention group], two recurrent stroke [one in intervention group before initiation of treatment, and one before group assignment had occurred]); all were unrelated to study participation. INTERPRETATION: 3 weeks of intensive speech and language therapy significantly enhanced verbal communication in people aged 70 years or younger with chronic aphasia after stroke, providing an effective evidence-based treatment approach in this population. Future studies should examine the minimum treatment intensity required for meaningful treatment effects, and determine whether treatment effects cumulate over repeated intervention periods. FUNDING: German Federal Ministry of Education and Research and the German Society for Aphasia Research and Treatment.

No Effect of Anodal Transcranial Direct Current Stimulation on Gamma-Aminobutyric Acid Levels in Patients with Recurrent Mild Traumatic Brain Injury.

In patients in the chronic phase after recurrent mild traumatic brain injury (mTBI), alterations in gamma-aminobutyric acid (GABA) concentration and receptor activity have been reported, possibly mediating subtle but persistent cognitive deficits and increased rate of dementia in older age. We evaluated whether anodal transcranial direct current stimulation (atDCS) over the primary motor cortex reduces GABA concentration and GABAB receptor activity in patients with recurrent mTBI. Seventeen patients (mean age 25, two women) in the chronic phase after recurrent mTBI and 22 healthy control subjects (mean age 26, two women) were included. All participants received comprehensive cognitive testing and detailed questionnaires on post-concussive symptoms at baseline. Subsequently, they participated in four experimental sessions, consisting of either magnetic resonance spectroscopy (MRS)/atDCS/MRS, transcranial magnetic stimulation (TMS)/atDCS/TMS, MRS/sham/MRS, or TMS/sham/TMS to determine GABA concentration (from MRS) and GABAB receptor activity (from TMS) after atDCS and after sham stimulation. Patients with mTBI scored significantly lower on verbal fluency tasks compared with healthy control subjects. GABA concentration at baseline was associated with the number of mTBI, although no group differences in GABA concentration and GABAB receptor activity were found. Moreover, no effects of atDCS on GABA concentration and receptor activity were seen in patients with mTBI or healthy control subjects. GABA concentration may increase with the number of mTBI, but atDCS did not modulate GABA concentration and receptor activity, as has been reported previously. Specifics of experimental design and analysis, but also characteristics of the respective samples, may account for these differential findings, and should be addressed in future larger studies.

Self-Assembled Active Plasmonic Waveguide with a Peptide-Based Thermomechanical Switch.

Nanoscale plasmonic waveguides composed of metallic nanoparticles are capable of guiding electromagnetic energy below the optical diffraction limit. Signal feed-in and readout typically require the utilization of electronic effects or near-field optical techniques, whereas for their fabrication mainly lithographic methods are employed. Here we developed a switchable plasmonic waveguide assembled from gold nanoparticles (AuNPs) on a DNA origami structure that facilitates a simple spectroscopic excitation and readout. The waveguide is specifically excited at one end by a fluorescent dye, and energy transfer is detected at the other end via the fluorescence of a second dye. The transfer distance is beyond the multicolor FRET range and below the Abbé limit. The transmittance of the waveguide can also be reversibly switched by changing the position of a AuNP within the waveguide, which is tethered to the origami platform by a thermoresponsive peptide. High-yield fabrication of the plasmonic waveguides in bulk was achieved using silica particles as solid supports. Our findings enable bulk solution applications for plasmonic waveguides as light-focusing and light-polarizing elements below the diffraction limit.

Long-range movement of large mechanically interlocked DNA nanostructures.

Interlocked molecules such as catenanes and rotaxanes, connected only via mechanical bonds have the ability to perform large-scale sliding and rotational movements, making them attractive components for the construction of artificial molecular machines and motors. We here demonstrate the realization of large, rigid rotaxane structures composed of DNA origami subunits. The structures can be easily modified to carry a molecular cargo or nanoparticles. By using multiple axle modules, rotaxane constructs are realized with axle lengths of up to 355 nm and a fuel/anti-fuel mechanism is employed to switch the rotaxanes between a mobile and a fixed state. We also create extended pseudo-rotaxanes, in which origami rings can slide along supramolecular DNA filaments over several hundreds of nanometres. The rings can be actively moved and tracked using atomic force microscopy.

cSPider - Evaluation of a Free and Open-Source Automated Tool to Analyze Corticomotor Silent Period.

BACKGROUND: The corticomotor silent period (CSP), as assessed noninvasively by transcranial magnetic stimulation (TMS) in the primary motor cortex, has been found to reflect intracortical inhibitory mechanisms. Analysis of CSP is mostly conducted manually. However, this approach is time-consuming, and comparison of results from different laboratories may be compromised by inter-rater variability in analysis. No open source program for automated analysis is currently available. METHODS/RESULTS: Here, we describe cross-validation with the manual analysis of an in-house written automated tool to assess CSP (cSPider). Results from automated routine were compared with results of the manual evaluation. We found high inter-method reliability between automated and manual analysis (p<0.001), and significantly reduced time for CSP analysis (median = 10.3 sec for automated analysis of 10 CSPs vs. median = 270 sec for manual analysis of 10 CSPs). cSPider can be downloaded free of charge. CONCLUSION: cSPider allows automated analysis of CSP in a reliable and time-efficient manner. Use of this open-source tool may help to improve comparison of data from different laboratories.

Effects of Different Analysis Strategies on Paired Associative Stimulation. A Pooled Data Analysis from Three Research Labs.

Paired associative stimulation (PAS) is a widely used transcranial magnetic stimulation (TMS) paradigm to non-invasively induce synaptic plasticity in the human brain in vivo. Altered PAS-induced plasticity has been demonstrated for several diseases. However, researchers are faced with a high inter- and intra-subject variability of the PAS response. Here, we pooled original data from nine PAS studies from three centers and analyzed the combined dataset of 190 healthy subjects with regard to age dependency, the role of stimulation parameters and the effect of different statistical methods. We observed no main effect of the PAS intervention over all studies (F(2;362) = 0.44; p = 0.644). The rate of subjects showing the expected increase of motor evoked potential (MEP) amplitudes was 53%. The PAS effect differed significantly between studies as shown by a significant interaction effect (F(16;362) = 1.77; p = 0.034) but post-hoc testing did not reveal significant effects after correction for multiple tests. There was a trend toward increased variability of the PAS effect in older subjects. Acquisition parameters differed across studies but without systematically influencing changes in MEP-size. The use of post/baseline quotients systematically indicated stronger PAS effects than post/baseline difference or the logarithm of the post/baseline quotient. The non-significant PAS effects across studies and a wide range of responder rates between studies indicate a high variability of this method. We were thus not able to replicate findings from a previous meta-analysis showing robust effects of PAS. No pattern emerged regarding acquisition parameters that at this point could guide future studies to reduce variability and help increase response rate. For future studies, we propose to report the responder rate and recommend the use of the logarithmized post/baseline quotient for further analyses to better address the possibility that results are driven by few extreme cases.

Cognitive function and brain structure after recurrent mild traumatic brain injuries in young-to-middle-aged adults.

Recurrent mild traumatic brain injuries (mTBIs) are regarded as an independent risk factor for developing dementia in later life. We here aimed to evaluate associations between recurrent mTBIs, cognition, and gray matter volume and microstructure as revealed by structural magnetic resonance imaging (MRI) in the chronic phase after mTBIs in young adulthood. We enrolled 20 young-to-middle-aged subjects, who reported two or more sports-related mTBIs, with the last mTBI > 6 months prior to study enrolment (mTBI group), and 21 age-, sex- and education matched controls with no history of mTBI (control group). All participants received comprehensive neuropsychological testing, and high resolution T1-weighted and diffusion tensor MRI in order to assess cortical thickness (CT) and microstructure, hippocampal volume, and ventricle size. Compared to the control group, subjects of the mTBI group presented with lower CT within the right temporal lobe and left insula using an a priori region of interest approach. Higher number of mTBIs was associated with lower CT in bilateral insula, right middle temporal gyrus and right entorhinal area. Our results suggest persistent detrimental effects of recurrent mTBIs on CT already in young-to-middle-aged adults. If additional structural deterioration occurs during aging, subtle neuropsychological decline may progress to clinically overt dementia earlier than in age-matched controls, a hypothesis to be assessed in future prospective trials.

Membrane-assisted growth of DNA origami nanostructure arrays.

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.