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 μM to 1 M NaCl), the applied electrode potential (−0.6 to +0.1 V vs Pt), and the temperature (1 to 50 °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.
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 ζpore and the protein zeta potential ζprotein 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, ζprotein − ζpore. 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.
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 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 (K
= 0.05 µM vs. 5 µM, t
= 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.
bsAb: bispecific antibody; FVIII/FIX/FX: coagulation factors VIII/IX/X; SPR: surface plasmon resonance; k
: association rate constant; k
: dissociation rate constant; K
: equilibrium dissociation constant; t
: dissociation half-life.
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.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.
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° 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 λ‐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.
Metallized nanopore structuresexhibit high potential for expanding the application scope of solid‐state nanopores as stochastic single‐molecule sensing devices. The fabrication of metallized SiN pores is presented, their noise performance is characterized, and real‐time translocation experiments of single DNA molecules are demonstrated.
We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural ...properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.
We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to ...extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10⁸ bound targets per cm² sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.
We report on the dynamic control over the orientation of short oligonucleotide strands which are tethered to gold surfaces in electrolyte solution. By applying alternating electrical bias potentials ...to the supporting electrodes we are able to induce a switching of the layer conformation between a “lying” and a “standing” state, simultaneously monitored in a contactless mode by fluorescence techniques. We demonstrate that our electrooptical experiments allow for an in-depth investigation of the intriguing molecular dynamics of DNA at surfaces and, moreover, how the dynamic response of these switchable biomolecular layers opens new prospects in label-free biosensing.
Relay race: The first example of a programmable DNA photonic wire is reported utilizing fluorophore‐tethered pyrrole‐imidazole polyamides for site‐directed fluorophore assembly along a pre‐formed DNA ...duplex (see scheme; PB=Pacific Blue, Cy3=Cyanine 3; orange rectangles=fluorophore). The importance of such control is revealed by efficient energy transport over distances in excess of 27 nm.
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