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.

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.

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.

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.

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.

DNA nanostructures interacting with lipid bilayer membranes.

CONSPECTUS: DNA has been previously shown to be useful as a material for the fabrication of static nanoscale objects, and also for the realization of dynamic molecular devices and machines. In many cases, nucleic acid assemblies directly mimic biological structures, for example, cytoskeletal filaments, enzyme scaffolds, or molecular motors, and many of the applications envisioned for such structures involve the study or imitation of biological processes, and even the interaction with living cells and organisms. An essential feature of biological systems is their elaborate structural organization and compartmentalization, and this most often involves membranous structures that are formed by dynamic assemblies of lipid molecules. Imitation of or interaction with biological systems using the tools of DNA nanotechnology thus ultimately and necessarily also involves interactions with lipid membrane structures, and thus the creation of DNA-lipid hybrid assemblies. Due to their differing chemical nature, however, highly charged nucleic acids and amphiphilic lipids do not seem the best match for the construction of such systems, and in fact they are rarely found in nature. In recent years, however, a large variety of lipid-interacting DNA conjugates were developed, which are now increasingly being applied also for the realization of DNA nanostructures interacting with lipid bilayer membranes. In this Account, we will present the current state of this emerging class of nanosystems. After a brief overview of the basic biophysical and biochemical properties of lipids and lipid bilayer membranes, we will discuss how DNA molecules can interact with lipid membranes through electrostatic interactions or via covalent modification with hydrophobic moieties. We will then show how such DNA-lipid interactions have been utilized for the realization of DNA nanostructures attached to or embedded within lipid bilayer membranes. Under certain conditions, DNA nanostructures remain mobile on membranes and can dynamically associate into higher order complexes. Hydrophobic modification of DNA nanostructures can further result in intra- or intermolecular aggregation, which can also be utilized as a structural switching mechanism. Appropriate design and chemical modification even allows insertion of DNA nanostructures into lipid bilayer membranes, resulting in artificial ion channel mimics made from DNA. Interactions of DNA nanodevices with living cells also involve interactions with membrane structures. DNA-based nanostructures can be directed to cell surfaces via antibody-antigen interactions, and their cellular uptake can be stimulated by modification with appropriate receptor ligands. In the future, membrane-embedded DNA nanostructures are expected to find application in diverse areas ranging from basic biological research over nanotechnology to synthetic biology.

Hydrophobic actuation of a DNA origami bilayer structure.

Amphiphilic compounds have a strong tendency to form aggregates in aqueous solutions. It is shown that such aggregation can be utilized to fold cholesterol-modified, single-layered DNA origami structures into sandwich-like bilayer structures, which hide the cholesterol modifications in their interior. The DNA bilayer structures unfold after addition of the surfactant Tween 80, and also in the presence of lipid bilayer membranes, with opening kinetics well described by stretched exponentials. It is also demonstrated that by combination with an appropriate lock and key mechanism, hydrophobic actuation of DNA sandwiches can be made conditional on the presence of an additional molecular input such as a specific DNA sequence.

Relationship between excitability, plasticity and thickness of the motor cortex in older adults.

The relationship between brain structure, cortical physiology, and learning ability in older adults is of particular interest in understanding mechanisms of age-related cognitive decline. Only a few studies addressed this issue so far, yielding mixed results. Here, we used comprehensive multiple regression analyses to investigate associations between brain structure on the one hand, i.e., cortical thickness (CT), fractional anisotropy (FA) of the pyramidal tract and individual coil-to-cortex distance, and cortical physiology on the other hand, i.e. motor cortex excitability and long-term potentiation (LTP)-like cortical plasticity, in healthy older adults (mean age 64 years, 14 women). Additional exploratory analyses assessed correlations between cortical physiology and learning ability in the verbal domain. In the regression models, we found that cortical excitability could be best predicted by CT of the hand knob of the primary motor cortex (CT-M1HAND) and individual coil-to-cortex distance, while LTP-like cortical plasticity was predicted by CT-M1HAND and FA of the pyramidal tract. Exploratory analyses revealed a significant inverse correlation between cortical excitability and learning ability. In conclusion, higher cortical excitability was associated with lower CT and lower learning ability in a cohort of healthy older adults, in line with previous reports of increased cortical excitability in patients with cortical atrophy and cognitive deficits due to Alzheimer's Disease. Cortical excitability may thus be a parameter to identify individuals at risk for cognitive decline and gray matter atrophy, a hypothesis to be explored in future longitudinal studies.

Cortical plasticity is preserved in nondemented older individuals with severe ischemic small vessel disease.

Ischemic small vessel disease (SVD) is a common finding on routine scans in older people, but cognitive sequelae vary considerably. To improve understanding of mechanisms underlying decline or preservation of cognitive function in this condition, we assessed cognition and cortical plasticity in 20 elderly subjects with severe SVD and 20 age-matched controls without SVD, as rated on conventional MRI. Cognitive status was determined with a neuropsychological test battery, cortical plasticity induced with a paired associative stimulation protocol. Microstructural white matter changes were further analyzed for fractional anisotrophy using diffusion tensor imaging. We found that cortical plasticity as well as memory functions were preserved in severe SVD, while executive functions showed trendwise or significant decreases. Within the SVD group, lower white matter integrity in parahippocampal regions and posterior parts of the corpus callosum was associated with larger cortical plasticity, an association not seen for prefrontal white matter tracts. Enhanced cortical plasticity in subjects with lower white matter integrity in memory-relevant areas might thus indicate a compensatory mechanism to counteract memory decline in severe SVD.

Biomarkers and perfusion--training-induced changes after stroke (BAPTISe): protocol of an observational study accompanying a randomized controlled trial.

BACKGROUND: Physical activity is believed to exert a beneficial effect on functional and cognitive rehabilitation of patients with stroke. Although studies have addressed the impact of physical exercise in cerebrovascular prevention and rehabilitation, the underlying mechanisms leading to improvement are poorly understood. Training-induced increase of cerebral perfusion is a possible mediating mechanism. Our exploratory study aims to investigate training-induced changes in blood biomarker levels and magnetic resonance imaging in patients with subacute ischemic stroke. METHODS/DESIGN: This biomarker-driven study uses an observational design to examine a subgroup of patients in the randomized, controlled PHYS-STROKE trial. In PHYS-STROKE, 215 patients with subacute stroke (hemorrhagic and ischemic) receive either 4 weeks of physical training (aerobic training, 5 times a week, for 50 minutes) or 4 weeks of relaxation sessions (5 times a week, for 50 minutes). A convenience sample of 100 of these patients with ischemic stroke will be included in BAPTISe and will receive magnetic resonance imaging (MRI) scans and an additional blood draw before and after the PHYS-STROKE intervention. Imaging scans will address parameters of cerebral perfusion, vessel size imaging, and microvessel density (the Q factor) to estimate the degree of neovascularization in the brain. Blood tests will determine several parameters of immunity, inflammation, endothelial function, and lipometabolism. Primary objective of this study is to evaluate differential changes in MRI and blood-derived biomarkers between groups. Other endpoints are next cerebrovascular events and functional status of the patient after the intervention and after 3 months assessed by functional scores, in particular walking speed and Barthel index (co-primary endpoints of PHYS-STROKE). Additionally, we will assess the association between functional outcomes and biomarkers including imaging results. For all endpoints we will compare changes between patients who received physical fitness training and patients who had relaxation sessions. DISCUSSION: This exploratory study will be the first to investigate the effects of physical fitness training in patients with ischemic stroke on MRI-based cerebral perfusion, pertinent blood biomarker levels, and functional outcome. The study may have an impact on current patient rehabilitation strategies and reveal important information about the roles of MRI and blood-derived biomarkers in ischemic stroke. TRIAL REGISTRATION: NCT01954797.

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.

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.

Enhanced rapid-onset cortical plasticity in CADASIL as a possible mechanism of preserved cognition.

Ischemic small vessel disease (SVD) may lead to cognitive impairment, but cognitive deficits with a given burden of SVD vary significantly. The underlying mechanisms of impaired or preserved cognition are unknown. Here, we investigated the impact of ischemic SVD on rapid-onset cortical plasticity, as induced with a paired-associative stimulation protocol. To exclude concomitant effects of aging, we examined 12 middle-aged patients (48.3 ± 8.3 years) with cerebral autosomal dominant arteriopathy with subcortical infarctions and leucoencephalopathy (CADASIL) who suffered from severe ischemic SVD and a group of 12 age-matched controls (49.9 ± 8.3 years). Cognitive status, motor performance and learning, and motor cortex excitability in response to cathodal transcranial direct current stimulation (ctDCS) were assessed. White matter integrity was analyzed by conventional magnetic resonance imaging and diffusion tensor imaging. We found that cognitive and motor functions were largely preserved in CADASIL patients, while rapid-onset cortical plasticity was significantly higher in the CADASIL group compared with controls (repeated measures analysis of variance [group × time] interaction: P = 0.03). This finding was even more pronounced in patients with higher white matter lesion load. ctDCS revealed no evidence of cortical dysplasticity. We conclude that increased rapid-onset cortical plasticity may contribute to largely preserved cognitive and motor function despite extensive ischemic SVD.

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.

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.

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.

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.