Assembly and Characterization of a Slingshot DNA Nanostructure for the Analysis of Bivalent and Bispecific Analytes with Biosensors.

The characterization of novel therapeutic antibodies with multivalent or multispecific binding sites requires new measurement modalities for biosensors, to discriminate the engagement of antigens via one, two, or even more binding moieties. The presentation of antigens on a sensor surface in a well-controlled spatial arrangement is a prerequisite for the successful interpretation of binding kinetics measurements of multivalent analytes, but the adjustment of defined distances between immobilized ligands is difficult to achieve in state-of-the-art biosensor systems. Here, we introduce a simple DNA nanostructure resembling a slingshot, which can be configured with two identical or two different antigens (bivalent or bispecific), which are spaced at a defined distance. We characterize the slingshot structure with a chip-based biosensor using electrically switchable DNA nanolevers and demonstrate that bivalent and monovalent antibodies selectively interact with slingshots that have been functionalized with two identical or two different antigens, respectively. The dissociation kinetics are quantified in real-time measurements and we show that the slingshot structure enables a clear differentiation between affinity and avidity effects.

The long zinc finger domain of PRDM9 forms a highly stable and long-lived complex with its DNA recognition sequence.

PR domain containing protein 9 (PRDM9) is a meiosis-specific, multi-domain protein that regulates the location of recombination hotspots by targeting its DNA recognition sequence for double-strand breaks (DSBs). PRDM9 specifically recognizes DNA via its tandem array of zinc fingers (ZnFs), epigenetically marks the local chromatin by its histone methyltransferase activity, and is an important tether that brings the DNA into contact with the recombination initiation machinery. A strong correlation between PRDM9-ZnF variants and specific DNA motifs at recombination hotspots has been reported; however, the binding specificity and kinetics of the ZnF domain are still obscure. Using two in vitro methods, gel mobility shift assays and switchSENSE, a quantitative biophysical approach that measures binding rates in real time, we determined that the PRDM9-ZnF domain forms a highly stable and long-lived complex with its recognition sequence, with a dissociation halftime of many hours. The ZnF domain exhibits an equilibrium dissociation constant (K D) in the nanomolar (nM) range, with polymorphisms in the recognition sequence directly affecting the binding affinity. We also determined that alternative sequences (15-16 nucleotides in length) can be specifically bound by different subsets of the ZnF domain, explaining the binding plasticity of PRDM9 for different sequences. Finally, longer binding targets are preferred than predicted from the numbers of ZnFs contacting the DNA. Functionally, a long-lived complex translates into an enzymatically active PRDM9 at specific DNA-binding sites throughout meiotic prophase I that might be relevant in stabilizing the components of the recombination machinery to a specific DNA target until DSBs are initiated by Spo11.

Electrical Actuation of a DNA Origami Nanolever on an Electrode.

Development of electrically powered DNA origami nanomachines requires effective means to actuate moving origami parts by externally applied electric fields. We demonstrate how origami nanolevers on an electrode can be manipulated (switched) at high frequency by alternating voltages. Orientation switching is long-time stable and can be induced by applying low voltages of 200 mV. The mechanical response time of a 100 nm long origami lever to an applied voltage step is less than 100 µs, allowing dynamic control of the induced motion. Moreover, through voltage assisted capture, origamis can be immobilized from folding solution without purification, even in the presence of excess staple strands. The results establish a way for interfacing and controlling DNA origamis with standard electronics, and enable their use as moving parts in electro-mechanical nanodevices.

Detection of the carcinogenic water pollutant benzo[a]pyrene with an electro-switchable biosurface.

The toxic nature of polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), neccessitates the monitoring of PAH contamination levels in food and the environment. Here we introduce an indirect immunoassay format using electro-switchable biosurfaces (ESB) for the detection of B[a]P in water. The association of anti-B[a]P antibodies to microelectrodes is analyzed in real-time by measuring changes in the oscillation dynamics of DNA nanolever probes, which are driven to switch their orientations by high-frequency electrical actuation. From the association kinetics, the active concentration of anti-B[a]P, and hence the B[a]P contamination of the sample, can be determined with picomolar sensitivity. The detection limit of the assay improves with measurement time because increasingly accurate analyses of the binding kinetics become possible. It is demonstrated that an exceedance of the permissible 10 ng/L (40 pM) limit for B[a]P is detectable in an unprecedented short assay time (<1 h), using a simple three-step workflow involving minimal sample preparation. The reproducibility was satisfying with standard deviations below 5%. Further, the utility of the assay for practical applications is exemplified by analyzing a river water sample.

Insight into the avidity-affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn.

Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of ß2-glycoprotein I (ß2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of ß2-GPI to the cardiolipin surface. Using the switchSENSE sensor, ß2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between ß2-GPI-dependent autoimmune- and ß2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Platform for in situ real-time measurement of protein-induced conformational changes of DNA.

A platform for in situ and real-time measurement of protein-induced conformational changes in dsDNA is presented. We combine electrical orientation of surface-bound dsDNA probes with an optical technique to measure the kinetics of DNA conformational changes. The sequence-specific Escherichia coli integration host factor is utilized to demonstrate protein-induced bending upon binding of integration host factor to dsDNA probes. The effects of probe surface density on binding/bending kinetics are investigated. The platform can accommodate individual spots of microarrayed dsDNA on individually controlled, lithographically designed electrodes, making it amenable for use as a high throughput assay.

Kinetic analysis of ternary and binary binding modes of the bispecific antibody emicizumab.

The binding properties of bispecific antibodies (bsAb) are crucial for their function, especially when two antigens are targeted on the same cell surface. Dynamic interactions between each of the antibody's arms and its cognate target cause the formation and decay of a biologically functional ternary complex. How association and dissociation processes work cooperatively, and how they influence the avidity of the ternary complex, is still poorly understood. Here, we present a biosensor assay for the simultaneous measurement of the binding kinetics of the therapeutic bsAb emicizumab (Hemlibra®) and its two targets, the blood coagulation factors IX and X (FIX, FX). We describe an automated workflow to characterize binary and ternary-binding modes, utilizing a Y-shaped DNA nanostructure to immobilize the antigens on a sensor and to emulate conditions on a cell or platelet surface by presenting the antigens with optimal accessibility for the bsAb flown over the sensor as analyte. We find that emicizumab binds FX much stronger than FIX (Kd = 0.05 µM vs. 5 µM, t1/2 = 20 s vs. 1 s) with profound consequences on the avidity of the ternary complex, which is dominated by FX's binding properties and a hand-off mechanism from FX to FIX. Moreover, formation and decay of the ternary complex depend on the bsAb concentration during the association phase. Emicizumab's in-vivo mode of action and the catalytic activation of FX can be rationalized from the analyzed binding kinetics. The assay and workflow are well suited for the screening of bispecific binders in drug discovery and provide valuable new kinetic information.Abbreviations: bsAb: bispecific antibody; FVIII/FIX/FX: coagulation factors VIII/IX/X; SPR: surface plasmon resonance; kon: association rate constant; koff: dissociation rate constant; KD: equilibrium dissociation constant; t1/2: dissociation half-life.

Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids.

The interaction of small molecules or proteins with RNA or DNA often involves changes in the nucleic acid (NA) folding and structure. A biophysical characterization of these processes helps us to understand the underlying molecular mechanisms. Here, we propose kinFRET (kinetics Förster resonance energy transfer), a real-time ensemble FRET methodology to measure binding and folding kinetics. With kinFRET, the kinetics of conformational changes of NAs (DNA or RNA) upon analyte binding can be directly followed via a FRET signal using a chip-based biosensor. We demonstrate the utility of this approach with two representative examples. First, we monitored the conformational changes of different formats of an aptamer (MN19) upon interaction with small-molecule analytes. Second, we characterized the binding kinetics of RNA recognition by tandem K homology (KH) domains of the human insulin-like growth factor II mRNA-binding protein 3 (IMP3), which reveals distinct kinetic contributions of the two KH domains. Our data demonstrate that kinFRET is well suited to study the kinetics and conformational changes of NA-analyte interactions.

Quantitation of affinity, avidity, and binding kinetics of protein analytes with a dynamically switchable biosurface.

A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K(D) in the pM regime), and the influence of competitive binders (EC(50) values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.

Photo-induced growth of DNA-capped silver nanoparticles.

We report the photo-induced nucleation and growth of silver nanoparticles in aqueous solution in the presence of DNA oligomers. An organic dye (Cy5) was used as a photosensitizer to initiate the nanoparticle growth upon illumination with 647 nm light. The formation of nanoparticles and growth kinetics were observed by extinction spectroscopy, dynamic light scattering, and transmission electron microscopy. Irradiation of the precursor solutions with light at the Cy5 absorption maximum triggered the instantaneous formation of spherical particles with a metallic core ~15 nm in diameter. Remarkably, the particles feature significantly larger effective hydrodynamic diameters (35 nm) in solution, indicative of a DNA ad-layer on the nanoparticle surface. Centrifugation experiments confirmed that DNA was inseparably associated with the nanoparticles and indicated that DNA oligomers adsorb onto the nanoparticle surface during growth, playing the role of a capping agent. The introduced method is a fast and facile way to prepare DNA-capped silver nanoparticles in a single growth step.

A pore-cavity-pore device to trap and investigate single nanoparticles and DNA molecules in a femtoliter compartment: confined diffusion and narrow escape.

Spatial confinement from the nano- to the microscale is ubiquitous in nature. Striving to understand the behavior of nanoscale objects in confined domains we present a nanofluidic silicon device which consists of two stacked nanopores forming the in/outlets to a pyramidal cavity of micrometer dimensions (10 fL volume). Being electrically addressable, charged objects can be actively loaded into, trapped inside, and unloaded from the "pore-cavity-pore" (PCP) device. When operated passively, confined Brownian motion and the entropy barriers of the nanopores govern the behavior of nano-objects within the PCP device. We present measurements with single fluorescent nanoparticles as well as particle-ensembles and analyze their trajectories and residence times. Experimental data are compared to random walk simulations and analytical theories on confined diffusion and the Brownian escape of nano-objects across entropy barriers. Single particle data corroborate analytical solutions of the narrow escape problem, but ensemble measurements indicate crowding effects even at low particle concentrations. The utilization of the device to trap biomolecules is demonstrated for single λ-DNA molecules.

Electrophoretic time-of-flight measurements of single DNA molecules with two stacked nanopores.

Electrophoretic transport through a solid-state nanodevice comprised of two stacked nanopore sensors is used to determine the free-solution mobility of DNA molecules based on their "time-of-flight" between the two pores. Mobility measurements are possible at very low (100 pM) DNA concentration and for low as well as high salt concentrations (here 30 mM and 1 M KCl). The mechanism of DNA transport through the device is elucidated by statistical analysis, showing the free-draining nature of the translocating DNA polymers and a barrier-dominated escape through the second pore. Furthermore, consecutive threading of single molecules through the two pores can be used to gain more detailed information on the dynamics of the molecules by correlation analysis, which also provides a direct electrical proof for translocation.

DNA origami gatekeepers for solid-state nanopores.

DNA has it covered: DNA origami gatekeeper nanoplates convert nanopores in solid-state membranes into versatile devices for label-free macromolecular sensing applications. The custom apertures in the nanoplates can be chemically addressed for sequence-specific detection of DNA.

Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis.

Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was found to be complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was observed. Here we study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential zeta(pore) and the protein zeta potential zeta(protein) are measured independently as a function of solution pH. Our results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, respectively; the resulting translocation direction can be predicted according to the difference in zeta potentials, zeta(protein) - zeta(pore). When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.

Multiplexed parallel single transport recordings on nanopore arrays.

We introduce a nanofabricated silicon chip for massively multiplexed analysis of membrane channels and transporters in suspended lipid membranes that does not require any surface modification or organic solvent. Transport processes through single membrane complexes are monitored by fluorescence. The chip consists of an array of well-defined nanopores, addressing an individual pyramidal back-reflecting 30-fL compartment. The setup allows simultaneous analyses of ∼1,000 single transmembrane events in one field of view, observing translocation kinetics of transmembrane complexes.

Conformations of end-tethered DNA molecules on gold surfaces: influences of applied electric potential, electrolyte screening, and temperature.

We describe the behavior of 72mer oligonucleotides that are end-tethered to gold surfaces under the influence of applied electric fields. The DNA extension is measured by fluorescence energy transfer as a function of the DNA hybridization state (single- and double-stranded), the concentration of monovalent salt in solution (100 microM to 1 M NaCl), the applied electrode potential (-0.6 to +0.1 V vs Pt), and the temperature (1 to 50 degrees C). At high ionic strength, the DNA conformations are very robust and independent of the applied electrode potential and temperature variations. In solutions of medium ionic strength, the DNA conformation can be manipulated efficiently by applying bias potentials to the Au electrodes. The molecules are repelled at negative potentials and attracted to the surface at positive potentials. The conformation transition occurs abruptly when the electrode bias is swept by merely 0.1 V across the transition potential, which shifts negatively when the salinity is decreased. The behavior can be understood by electrostatic screening arguments and, in the case of single-stranded DNA, when secondary structures are taken into account. At low ionic strength, the experiments reveal an intriguing temperature-dependent stiffening of single-stranded DNA, which can be rationalized by combining counterion condensation theory with the Odjik-Skolnick-Fixman description of the electrostatic persistence length and the unstacking of bases at elevated temperatures.

Fabrication of metallized nanopores in silicon nitride membranes for single-molecule sensing.

The fabrication and characterization of a metallized nanopore structure for the sensing of single molecules is described. Pores of varying diameters (>10 nm) are patterned into free-standing silicon nitride membranes by electron-beam lithography and reactive ion etching. Structural characterization by transmission electron microscopy (TEM) and tomography reveals a conical pore shape with a 40 degrees aperture. Metal films of Ti/Au are vapor deposited and the pore shape and shrinking are studied as a function of evaporated film thickness. TEM tomography analysis confirms metalization of the inner pore walls as well as conservation of the conical pore shape. In electrical measurements of the transpore current in aqueous electrolyte solution, the pores feature very low noise. The applicability of the metallized pores for stochastic sensing is demonstrated in real-time translocation experiments of single lambda-DNA molecules. We observe exceptionally long-lasting current blockades with a fine structure of distinct current levels, suggesting an attractive interaction between the DNA and the PEGylated metallic pore walls.

Detection and size analysis of proteins with switchable DNA layers.

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies.

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10-3 s-1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.