Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers.

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.

Controlled translocation of DNA through nanopores in carbon nano-, silicon-nitride- and lipid-coated membranes.

We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA molecule and the force dependence on the membrane thickness. The additional measurements presented in this paper further support our model.

Rational design of a cytotoxic dinuclear Cu2 complex that binds by molecular recognition at two neighboring phosphates of the DNA backbone.

The mechanism of the cytotoxic function of cisplatin and related anticancer drugs is based on their binding to the nucleobases of DNA. The development of new classes of anticancer drugs requires establishing other binding modes. Therefore, we performed a rational design for complexes that target two neighboring phosphates of the DNA backbone by molecular recognition resulting in a family of dinuclear complexes based on 2,7-disubstituted 1,8-naphthalenediol. This rigid backbone preorganizes the two metal ions for molecular recognition at the distance of two neighboring phosphates in DNA of 6-7 Å. Additionally, bulky chelating pendant arms in the 2,7-position impede nucleobase complexation by steric hindrance. We successfully synthesized the Cu(II)2 complex of the designed family of dinuclear complexes and studied its binding to dsDNA by independent ensemble and single-molecule methods like gel electrophoresis, precipitation, and titration experiments followed by UV-vis spectroscopy, atomic force microscopy (AFM), as well as optical tweezers (OT) and magnetic tweezers (MT) DNA stretching. The observed irreversible binding of our dinuclear Cu(II)2 complex to dsDNA leads to a blocking of DNA synthesis as studied by polymerase chain reactions and cytotoxicity for human cancer cells.

Hydrodynamic slip on DNA observed by optical tweezers-controlled translocation experiments with solid-state and lipid-coated nanopores.

We use optical tweezers to investigate the threading force on a single dsDNA molecule inside silicon-nitride nanopores between 6 and 70 nm in diameter, as well as lipid-coated solid-state nanopores. We observe a strong increase of the threading force for decreasing nanopore size that can be attributed to a significant reduction in the electroosmotic flow (EOF), which opposes the electrophoresis. Additionally, we show that the EOF can also be reduced by coating the nanopore wall with an electrically neutral lipid bilayer, resulting in an 85% increase in threading force. All experimental findings can be described by a quantitative theoretical model that incorporates a hydrodynamic slip effect on the DNA surface with a slip length of 0.5 nm.

Nanopore translocation dynamics of a single DNA-bound protein.

We study the translocation dynamics of a single protein molecule attached to a double-stranded DNA that is threaded through a solid-state nanopore by optical tweezers and an electric field (nanopore force spectroscopy). We find distinct asymmetric and retarded force signals that depend on the protein charge, the DNA elasticity and its counterionic screening in the buffer. A theoretical model where an isolated charge on an elastic, polyelectrolyte strand is experiencing an anharmonic nanopore potential was developed. Its results compare very well with the measured force curves and explain the experimental findings that the force depends linearly on the applied electric field and exhibits a small hysteresis during back and forth translocation cycles. Moreover, the translocation dynamics reflects the stochastic nature of the thermally activated hopping between two adjacent states in the nanopore that can be adequately described by Kramers rate theory.

Binding kinetics of bisintercalator Triostin a with optical tweezers force mechanics.

The binding kinetics of the intercalative binding of Triostin A to lambda-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.

Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores.

We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference.

Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications.

Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications.

Video-based and interference-free axial force detection and analysis for optical tweezers.

For measuring the minute forces exerted on single molecules during controlled translocation through nanopores with sub-piconewton precision, we have developed a video-based axial force detection and analysis system for optical tweezers. Since our detection system is equipped with a standard and versatile CCD video camera with a limited bandwidth offering operation at moderate light illumination with minimal sample heating, we integrated Allan variance analysis for trap stiffness calibration. Upon manipulating a microbead in the vicinity of a weakly reflecting surface with simultaneous axial force detection, interference effects have to be considered and minimized. We measured and analyzed the backscattering light properties of polystyrene and silica microbeads with different diameters and propose distinct and optimized experimental configurations (microbead material and diameter) for minimal light backscattering and virtually interference-free microbead position detection. As a proof of principle, we investigated the nanopore threading forces of a single dsDNA strand attached to a microbead with an overall force resolution of ±0.5 pN at a sample rate of 123 Hz.

Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers.

We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

Molecular mechanisms and kinetics between DNA and DNA binding ligands.

Mechanical properties of single double-stranded DNA (dsDNA) in the presence of different binding ligands were analyzed in optical-tweezers experiments with subpiconewton force resolution. The binding of ligands to DNA changes the overall mechanic response of the dsDNA molecule. This fundamental property can be used for discrimination and identification of different binding modes and, furthermore, may be relevant for various processes like nucleosome packing or applications like cancer therapy. We compared the effects of the minor groove binder distamycin-A, a major groove binding alpha-helical peptide, the intercalators ethidium bromide, YO-1, and daunomycin as well as the bisintercalator YOYO-1 on lambda-DNA. Binding of molecules to the minor and major groove of dsDNA induces distinct changes in the molecular elasticity compared to the free dsDNA detectable as a shift of the overstretching transition to higher forces. Intercalating molecules affect the molecular mechanics by a complete disappearance of the B-S transition and an associated increase in molecular contour length. Significant force hysteresis effects occurring during stretching/relaxation cycles with velocities >10 nm/s for YOYO-1 and >1000 nm/s for daunomycin. These indicate structural changes in the timescale of minutes for the YOYO-DNA and of seconds for the daunomycin-DNA complexes, respectively.