Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays.

Molecular and Thermodynamic Factors Explain the Passivation Properties of Poly(ethylene glycol)-Coated Substrate Surfaces against Fluorophore-Labeled DNA Oligonucleotides.

Poly(ethylene glycol) (PEG) nanofilms are used to avert the nonspecific binding of biomolecules on substrate surfaces in biomedicine and bioanalysis including modern fluorescence-based DNA sensing and sequencing chips. A fundamental and coherent understanding of the interactions between fluorophore-tagged DNA, PEG-films, and substrates in terms of molecular and energetic factors is, however, missing. Here we explore a large parameter space to elucidate how PEG layers passivate metal oxide surfaces against Cy3-labeled DNA probes. The driving force for probe adsorption is found to be the affinity of the fluorophore to the substrate, while the high-quality PEG films prevent adsorption to bare ITO surfaces. The amount of nonrepelled, surface-bound DNA strongly depends on oligonucleotide size, PEG chain length, and incubation temperature. To explain these observations, we develop an experimentally validated theory to provide a microscopic picture of the PEG layer and show that adsorbed DNA molecules reside within the film by end-tethering the fluorophore to the ITO surface. To compensate for the local accumulation of negatively charged DNA, counterions condense on the adsorbed probes within the layer. The model furthermore explains that surface passivation is governed by the interdependence of molecular size, conformation, charge, ion condensation, and environmental conditions. We finally report for the first time on the detailed thermodynamic values that show how adsorption results from a balance between large opposing energetic factors. The insight of our study can be applied to rationally engineer PEG nanolayers for improved functional performance in DNA analysis schemes and may be expanded to other polymeric thin films.

A Simplified and Robust Activation Procedure of Glass Surfaces for Printing Proteins and Subcellular Micropatterning Experiments.

Depositing biomolecule micropatterns on solid substrates via microcontact printing (µCP) usually requires complex chemical substrate modifications to initially create reactive surface groups. Here, we present a simplified activation procedure for untreated solid substrates based on a commercial polymer metal ion coating (AnteoBindTM Biosensor reagent) that allows for direct µCP and the strong attachment of proteins via avidity binding. In proof-of-concept experiments, we identified the optimum working concentrations of the surface coating, characterized the specificity of protein binding and demonstrated the suitability of this approach by subcellular micropatterning experiments in living cells. Altogether, this method represents a significant enhancement and simplification of existing µCP procedures and further increases the accessibility of protein micropatterning for cell biological research questions.

Microcontact Printing of Biomolecules on Various Polymeric Substrates: Limitations and Applicability for Fluorescence Microscopy and Subcellular Micropatterning Assays.

Polymeric materials play an emerging role in biosensing interfaces. Within this regard, polymers can serve as a superior surface for binding and printing of biomolecules. In this study, we characterized 11 different polymer foils [cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), DI-Acetate, Lumirror 4001, Melinex 506, Melinex ST 504, polyamide 6, polyethersulfone, polyether ether ketone, and polyimide] to test for the applicability for surface functionalization, biomolecule micropatterning, and fluorescence microscopy approaches. Pristine polymer foils were characterized via UV-vis spectroscopy. Functional groups were introduced by plasma activation and epoxysilane-coating. Polymer modification was evaluated by water contact angle measurement and X-ray photoelectron spectroscopy. Protein micropatterns were fabricated using microcontact printing. Functionalized substrates were characterized via fluorescence contrast measurements using epifluorescence and total internal reflection fluorescence microscopy. Results showed that all polymer substrates could be chemically modified with epoxide functional groups, as indicated by reduced water contact angles compared to untreated surfaces. However, transmission and refractive index measurements revealed differences in important optical parameters, which was further proved by fluorescence contrast measurements of printed biomolecules. COC, COP, and PMMA were identified as the most promising alternatives to commonly used glass coverslips, which also showed superior applicability in subcellular micropatterning experiments.

Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models.

SCOPE: Sea buckthorn (Hippophaes rhamnoides) is capable of ameliorating disturbed glucose metabolism in animal models and human subjects. Here, the effect of sea buckthorn oil as well as of extracts of fruits, leaves, and press cake on postprandial glucose metabolism is systematically investigated. METHODS AND RESULTS: Sea buckthorn did neither exert decisive effects in an in vitro model of intestinal glucose absorption nor did it alter insulin secretion. However, sea buckthorn stimulates GLUT4 translocation to the plasma membrane comparable to insulin, indicative of increased glucose clearance from the circulation. Isorhamnetin is identified in all sea buckthorn samples investigated and is biologically active in triggering GLUT4 cell surface localization. Consistently, sea buckthorn products lower circulating glucose by ≈10% in a chick embryo model. Moreover, sea buckthorn products fully revert hyperglycemia in the nematode Caenorhabditis elegans while they are ineffective in Drosophila melanogaster under euglycemic conditions. CONCLUSION: These data indicate that edible sea buckthorn products as well as by-products are promising resources for hypoglycemic nutrient supplements that increase cellular glucose clearance into target tissues.

Subcellular Dynamic Immunopatterning of Cytosolic Protein Complexes on Microstructured Polymer Substrates.

Analysis of protein-protein interactions in living cells by protein micropatterning is currently limited to the spatial arrangement of transmembrane proteins and their corresponding downstream molecules. Here, we present a robust and straightforward method for dynamic immunopatterning of cytosolic protein complexes by use of an artificial transmembrane bait construct in combination with microstructured antibody arrays on cyclic olefin polymer substrates. As a proof, the method was used to characterize Grb2-mediated signaling pathways downstream of the epidermal growth factor receptor (EGFR). Ternary protein complexes (Shc1:Grb2:SOS1 and Grb2:Gab1:PI3K) were identified, and we found that EGFR downstream signaling is based on constitutively bound (Grb2:SOS1 and Grb2:Gab1) as well as on agonist-dependent protein associations with transient interaction properties (Grb2:Shc1 and Grb2:PI3K). Spatiotemporal analysis further revealed significant differences in stability and exchange kinetics of protein interactions. Furthermore, we could show that this approach is well suited to study the efficacy and specificity of SH2 and SH3 protein domain inhibitors in a live cell context. Altogether, this method represents a significant enhancement of quantitative subcellular micropatterning approaches as an alternative to standard biochemical analyses.

A High-Content Screen for the Identification of Plant Extracts with Insulin Secretion-Modulating Activity.

Bioactive plant compounds and extracts are of special interest for the development of pharmaceuticals. Here, we describe the screening of more than 1100 aqueous plant extracts and synthetic reference compounds for their ability to stimulate or inhibit insulin secretion. To quantify insulin secretion in living MIN6 ß cells, an insulin-Gaussia luciferase (Ins-GLuc) biosensor was used. Positive hits included extracts from Quillaja saponaria, Anagallis arvensis, Sapindus mukorossi, Gleditsia sinensis and Albizia julibrissin, which were identified as insulin secretion stimulators, whereas extracts of Acacia catechu, Myrtus communis, Actaea spicata L., Vaccinium vitis-idaea and Calendula officinalis were found to exhibit insulin secretion inhibitory properties. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to characterize several bioactive compounds in the selected plant extracts, and these bioactives were retested for their insulin-modulating properties. Overall, we identified several plant extracts and some of their bioactive compounds that may be used to manipulate pancreatic insulin secretion.

Fabrication, Characterization and Application of Biomolecule Micropatterns on Cyclic Olefin Polymer (COP) Surfaces with Adjustable Contrast.

Peptide and protein micropatterns are powerful tools for the investigation of various cellular processes, including protein-protein interactions (PPIs). Within recent years, various approaches for the production of functional surfaces have been developed. Most of these systems use glass as a substrate, which has several drawbacks, including high fragility and costs, especially if implemented for fluorescence microscopy. In addition, conventional fabrication technologies such as microcontact printing (µCP) are frequently used for the transfer of biomolecules to the glass surface. In this case, it is challenging to adjust the biomolecule density. Here, we show that cyclic olefin polymer (COP) foils, with their encouraging properties, including the ease of manufacturing, chemical resistance, biocompatibility, low water absorption, and optical clarity, are a promising alternative to glass substrates for the fabrication of micropatterns. Using a photolithography-based approach, we generated streptavidin/biotinylated antibody patterns on COPs with the possibility of adjusting the pattern contrast by varying plasma activation parameters. Our experimental setup was finally successfully implemented for the analysis of PPIs in the membranes of live cells via total internal reflection fluorescence (TIRF) microscopy.

Arrays of Individual DNA Molecules on Nanopatterned Substrates.

Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.

Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.

Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged ß3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment.