Exploring the Synergies of Single‐Molecule Fluorescence and 2D Materials Coupled by DNA

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided

High force catch bond mechanism of bacterial adhesion in the human gut

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at similar to 500 pN and the other at similar to 200 pN at loading rates from 1-100 nN s(-1). A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds

Single antibody detection in a DNA origami nanoantenna

DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm

Programming Light-Harvesting Efficiency Using DNA Origami.

The remarkable performance and quantum efficiency of biological light-harvesting complexes has prompted a multidisciplinary interest in engineering biologically inspired antenna systems as a possible route to novel solar cell technologies. Key to the effectiveness of biological "nanomachines" in light capture and energy transport is their highly ordered nanoscale architecture of photoactive molecules. Recently, DNA origami has emerged as a powerful tool for organizing multiple chromophores with base-pair accuracy and full geometric freedom. Here, we present a programmable antenna array on a DNA origami platform that enables the implementation of rationally designed antenna structures. We systematically analyze the light-harvesting efficiency with respect to number of donors and interdye distances of a ring-like antenna using ensemble and single-molecule fluorescence spectroscopy and detailed Förster modeling. This comprehensive study demonstrates exquisite and reliable structural control over multichromophoric geometries and points to DNA origami as highly versatile platform for testing design concepts in artificial light-harvesting networks.A. W. C. acknowledges support from the Winton Programme for the Physics of Sustainability. U. F. K. was partly supported by an ERC starting grant (PassMembrane, EY 261101). E. A.H. acknowledges support from Janggen-Pöhn Stiftung and the Schweizerischer Nationalfonds (SNF). P. T. acknowledges support by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC). B. W. gratefully acknowledges support by the Braunschweig International Graduate School of Metrology B-IGSM and the DFG Research Training Group GrK1952/1 ‘Metrology for Complex Nanosystems’. P. M. thankfully acknowledges the support of the EPSRC Centre for Doctoral Training in Sensor Technologies and Applications EP/L015889/1.This is the final version of the article. It first appeared from ACS via https://doi.org/10.1021/acs.nanolett.5b0513

How Blinking Affects Photon Correlations in Multichromophoric Nanoparticles

A single chromophore can only emit a maximum of one single photon per excitation cycle. This limitation results in a phenomenon commonly referred to as photon antibunching (pAB). When multiple chromophores contribute to the fluorescence measured, the degree of pAB has been used as a metric to “count” the number of chromophores. But the fact that chromophores can switch randomly between bright and dark states, also impacts pAB and can lead to incorrect chromophore numbers being determined from pAB measurements. By both simulations and experiment, we demonstrate how pAB is affected by independent and collective chromophore blinking, enabling us to formulate universal guidelines for correct interpretation of pAB measurements. We use DNA-origami nanostructures to design multichromophoric model systems that exhibit either independent or collective chromophore blinking. Two approaches are presented that can distinguish experimentally between these two blinking mechanisms. The first one utilizes the different excitation intensity dependence on the blinking mechanisms. The second approach exploits the fact that collective blinking implies energy transfer to a quenching moiety, which is a time-dependent process. In pulsed-excitation experiments, the degree of collective blinking can therefore be altered by time gating the fluorescence photon stream, enabling us to extract the energy-transfer rate to a quencher. The ability to distinguish between different blinking mechanisms is valuable in materials science, such as for multichromophoric nanoparticles like conjugated-polymer chains, as well as in biophysics, e.g. for quantitative analysis of protein assemblies by counting chromophores

Excitonic AND Logic Gates on DNA Brick Nanobreadboards

A promising application of DNA self-assembly is the fabrication of chromophore-based excitonic devices. DNA brick assembly is a compelling method for creating programmable nanobreadboards on which chromophores may be rapidly and easily repositioned to prototype new excitonic devices, optimize device operation, and induce reversible switching. Using DNA nanobreadboards, we have demonstrated each of these functions through the construction and operation of two different excitonic AND logic gates. The modularity and high chromophore density achievable via this brick-based approach provide a viable path toward developing information processing and storage systems

Heterogeneous Pattern of Retinal Nerve Fiber Layer in Multiple Sclerosis. High Resolution Optical Coherence Tomography: Potential and Limitations

BACKGROUND: Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique. METHODOLOGY: Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP). PRINCIPAL FINDINGS: All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9-540 months). CONCLUSIONS: RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels)

Localized direction selective responses in the dendrites of visual interneurons of the fly

<p>Abstract</p> <p>Background</p> <p>The various tasks of visual systems, including course control, collision avoidance and the detection of small objects, require at the neuronal level the dendritic integration and subsequent processing of many spatially distributed visual motion inputs. While much is known about the pooled output in these systems, as in the medial superior temporal cortex of monkeys or in the lobula plate of the insect visual system, the motion tuning of the elements that provide the input has yet received little attention. In order to visualize the motion tuning of these inputs we examined the dendritic activation patterns of neurons that are selective for the characteristic patterns of wide-field motion, the lobula-plate tangential cells (LPTCs) of the blowfly. These neurons are known to sample direction-selective motion information from large parts of the visual field and combine these signals into axonal and dendro-dendritic outputs.</p> <p>Results</p> <p>Fluorescence imaging of intracellular calcium concentration allowed us to take a direct look at the local dendritic activity and the resulting local preferred directions in LPTC dendrites during activation by wide-field motion in different directions. These 'calcium response fields' resembled a retinotopic dendritic map of local preferred directions in the receptive field, the layout of which is a distinguishing feature of different LPTCs.</p> <p>Conclusions</p> <p>Our study reveals how neurons acquire selectivity for distinct visual motion patterns by dendritic integration of the local inputs with different preferred directions. With their spatial layout of directional responses, the dendrites of the LPTCs we investigated thus served as matched filters for wide-field motion patterns.</p

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups