Rainer Eckel

Identification of Binding Mechanisms in Single Molecule–DNA Complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.

Compact microscope-based optical tweezers system for molecular manipulation

A compact single beam optical tweezers system for force measurements and manipulation of individual double-stranded deoxyribonucleic acid (DNA) molecules was integrated into a commercial inverted optical microscope. A maximal force of 150 pN combined with a force sensitivity of less than 0.5 pN allows measurements of elastic properties of single molecules which complements and overlaps the force regime accessible with atomic force microscopy (AFM). The manipulation and measurement performance of this system was tested with individual λ-DNA molecules and renders new aspects of dynamic forces phenomena with higher precision in contrast to AFM studies. An integrated liquid handling system with a fluid cell allows investigation of the force response of individual DNA molecules in the presence of DNA binding agents. Comparison of YOYO-1-, ethidium bromide intercalated DNA, and distamycin-A complexed DNA revealed accurate and reproducible differences in the force response to an external load. This opens the possibility to use it as a single molecule biosensor to investigate DNA binding agents and even to identify molecular binding mechanisms.

Single-Molecule Experiments to Elucidate the Minimal Requirement for DNA Recognition by Transcription Factor Epitopes

Interactions between proteins and DNA are essential for the regulation of cellular processes in all living organisms. In this context, it is of special interest to investigate the sequence-specific molecular recognition between transcription factors and their cognate DNA sequences. As a model system, peptide and protein epitopes of the DNA-binding domain (DBD) of the transcription factor PhoB from Escherichia coli are analyzed with respect to DNA binding at the single-molecule level. Peptides representing the amphiphilic recognition helix of the PhoB DBD (amino acids 190-209) are chemically synthesized and C-terminally modified with a linker for atomic force microscopy-dynamic force spectroscopy experiments (AFM-DFS). For comparison, the entire PhoB DBD is overexpressed in E. coli and purified using an intein-mediated protein purification method. To facilitate immobilization for AFM-DFS experiments, an additional cysteine residue is ligated to the protein. Quantitative AFM-DFS analysis proves the specificity of the interaction and yields force-related properties and kinetic data, such as thermal dissociation rate constants. An alanine scan for strategic residues in both peptide and protein sequences is performed to reveal the contributions of single amino acid residues to the molecular-recognition process. Additionally, DNA binding is substantiated by electrophoretic mobility-shift experiments. Structural differences of the peptides, proteins, and DNA upon complex formation are analyzed by circular dichroism spectroscopy. This combination of techniques eventually provides a concise picture of the contribution of epitopes or single amino acids in PhoB to DNA binding.

On the way to supramolecular photochemistry at the single-molecule level

Two examples of artificial supramolecular host-guest systems derived from resorc[4]arenes (calix[n]arenes based on resorcinol) and ammonium ions as guests have been studied by atomic force microscopy (AFM). For the first time, real single-molecule events have been determined for this type of supramolecular complexes and off-rates as well as molecular parameters of single-molecule aggregates such as the depths of the binding pocket (molecular length parameter) could be measured by applying the methods of dynamic force spectroscopy. In addition, this technique was also applied to differentiate between the two states (open and closed) of a photoswitchable resorc[4]arene-anthracene tweezer. An investigation of the exchange rates of various complexes in the gas phase by means of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry confirmed the results of the AFM study.

Combined TIRF-AFM Setup: Controlled Quenching of Individual Quantum Dots

Single molecules can nowadays be investigated by means of optical, mechanical and electrical methods. Fluorescence imaging and spectroscopy yield valuable and quantitative information about the optical properties and the spatial distribution of single molecules. Force spectroscopy by atomic force microscopy (AFM) or optical tweezers allows addressing, manipulation and quantitative probing of the nanomechanical properties of individual macromolecules. We present a combined AFM and total internal reflection fluorescence (TIRF) microscopy setup that enables ultrasensitive laser induced fluorescence detection of individual fluorophores, control of the AFM probe position in x, y and z-direction with nanometer precision, and simultaneous investigation of optical and mechanical properties at the single molecule level. Here, we present the distance-controlled quenching of semiconductor quantum dot clusters with an AFM tip. In future applications, fluorescence resonant energy transfer between single donor and acceptor molecules will be investigated.