Correction to: Binding mechanism of anti-cancer chemotherapeutic drug mitoxantrone to DNA characterized by magnetic tweezers.

Following publication of this article [1] we found a typographical error in the results reported in the abstract. The corrected sentences should read as below.

Binding mechanism of anti-cancer chemotherapeutic drug mitoxantrone to DNA characterized by magnetic tweezers.

BACKGROUND: Chemotherapeutic agents (anti-cancer drugs) are small cytostatic or cytotoxic molecules that often bind to double-stranded DNA (dsDNA) resulting in modifications of their structural and nanomechanical properties and thus interfering with the cell proliferation process. METHODS: We investigated the anthraquinone compound mitoxantrone that is used for treating certain cancer types like leukemia and lymphoma with magnetic tweezers as a single molecule nanosensor. In order to study the association of mitoxantrone with dsDNA, we conducted force-extension and mechanical overwinding experiments with a sensitivity of 10-14 N. RESULTS: Using this method, we were able to estimate an equilibrium constant of association Ka ≈ 1 × 105 M-1 as well as a binding site size of n ≈ 2.5 base pairs for mitoxantrone. An unwinding angle of mitoxantrone-intercalation of ϑ ≈ 16° was determined. CONCLUSION: Moreover, we observed a complex concentration-dependent bimodal binding behavior, where mitoxantrone associates to dsDNA as an intercalator and groove binder simultaneously at low concentrations and as a mere intercalator at high concentrations.

Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy.

Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously.

Systems nanobiology: from quantitative single molecule biophysics to microfluidic-based single cell analysis.

Detailed and quantitative information about structure-function relation, concentrations and interaction kinetics of biological molecules and subcellular components is a key prerequisite to understand and model cellular organisation and temporal dynamics. In systems nanobi-ology, cellular processes are quantitatively investigated at the sensitivity level of single molecules and cells. This approach provides direct access to biomolecular information without being statistically ensemble-averaged, their associated distribution functions, and possible subpopulations. Moreover at the single cell level, the interplay of regulated genomic information and proteomic variabilities can be investigated and attributed to functional peculiarities. These requirements necessitate the development of novel and ultrasensitive methods and instruments for single molecule detection, microscopy and spectroscopy for analysis without the need of amplification and preconcentration. In this chapter, we present three methodological applications that demonstrate how quantitative informations can be accessed that are representative for cellular processes or single cell analysis like gene expression regulation, intracellular protein translocation dynamics, and single cell protein fingerprinting. First, the interaction kinetics of transcriptionally regulated DNA-protein interaction can be quantitatively investigated with single molecule force spectroscopy allowing a molecular affinity ranking. Second, intracellular protein dynamics for a transcription regulator migrating form the nucleus to the cytoplasm can be quantitatively monitored by photoactivable GFP and two-photon laser scanning microscopy. And third, a microfluidic-based method for label-free single cell proteomics and fingerprinting and first label-free single cell electropherograms are presented which include the manipulation and steering of single cells in a microfluidic device.

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time.

We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We determined average time constants of 51 s and 125 s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively.

Adsorption of DNA and electric fields decrease the rigidity of lipid vesicle membranes.

The adsorption of calf-thymus DNA-fragments of 300 +/- 50 base pairs (bp) to the outer membrane monolayer of unilamellar lipid vesicles in the presence of Ca2+ ions has been quantified by the standard method of chemical relaxation spectrometry using polarized light. The vesicles of radius a = 150 +/- 45 nm are prepared from bovine brain extract type III containing 80-85% phosphatidylserine (PS) and palmitoyl-oleoyl-phosphatidylcholine (POPC) in the molar ratio PS : 2POPC; total lipid concentration [L(t)] = 1 mM in 1 mM HEPES buffer, pH 7.4 at T = 293 K (20 degrees C). The turbidity relaxations of vesicle suspensions, at the wavelength lambda = 365 nm at two characteristic electric field strengths are identified as electroelongation of the whole vesicle coupled to smoothing of thermal membrane undulations and membrane stretching, and at higher fields, to membrane electroporation (MEP). The elongation kinetics indicates that the DNA adsorption renders the membrane more flexible and prone to membrane electroporation (MEP). Remarkably, it is found that the Ca-mediated adsorption of DNA (D) decreases both, bending rigidity kappa and stretching modulus K, along an unique Langmuir adsorption isotherm for the fraction of bound DNA at the given Ca concentration [Ca(t)] = 0.25 mM. The characteristic chemo-mechanical parameter of the isotherm is the apparent dissociation equilibrium constant K(D,Ca) = 100 +/- 10 microM (bp) of the ternary complex DCaB of DNA base pairs (bp) and Ca binding to sites B on the outer vesicle surface. Whereas both kappa and K decrease in the presence of high electric fields (E), the key parameter K(D,Ca) is independent of E in the range 0 < or = E/(kV cm(-1)) < or = 40.

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.