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.

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.

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.

Magnesium-Dependent Electrical Actuation and Stability of DNA Origami Rods.

Dynamic methods of biosensing based on electrical actuation of surface-tethered nanolevers require the use of levers whose movement in ionic liquids is well controllable and stable. In particular, mechanical integrity of the nanolevers in a wide range of ionic strengths will enable to meet the chemical conditions of a large variety of applications where the specific binding of biomolecular analytes is analyzed. Herein, we study the electrically induced switching behavior of different rodlike DNA origami nanolevers and compare to the actuation of simply double-stranded DNA nanolevers. Our measurements reveal a significantly stronger response of the DNA origami to switching of electrode potential, leading to a smaller potential change necessary to actuate the origami and subsequently to a long-term stable movement. Dynamic measurements in buffer solutions with different Mg2+ contents show that the levers do not disintegrate even at very low ion concentrations and constant switching stress and thus provide stable actuation performance. The latter will pave the way for many new applications without largely restricting application-specific environments.

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.

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.

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.

Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces.

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

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.

Protein analysis by time-resolved measurements with an electro-switchable DNA chip.

Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements.

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.

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.

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.

Stochastic sensing of proteins with receptor-modified solid-state nanopores.

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

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.

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.

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.

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.