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We demonstrate a single DNA molecule optical mapping assay able to resolve a specific Escherichia coli strain from other strains. The assay is based on competitive binding of the fluorescent dye YOYO-1 and the AT-specific antibiotic netropsin. The optical map is visualized by stretching the DNA molecules in nanofluidic channels. We optimize the experimental conditions to obtain reproducible barcodes containing as much information as possible. We implement a multi-ligand transfer matrix method for calculating theoretical barcodes from known DNA sequences. Our method extends previous theoretical approaches for competitive binding of two types of ligands to many types of ligands and introduces a recursive approach that allows long barcodes to be calculated with standard computer floating point formats. The identification of a specific E. coli strain (CCUG 10979) is based on mapping of 50-160 kilobasepair experimental DNA fragments onto the theoretical genome using the developed theory. Our identification protocol introduces two theoretical constructs: a P-value for a best experiment-theory match and an information score threshold. The developed methods provide a novel optical mapping toolbox for identification of bacterial species and strains. The protocol does not require cultivation of bacteria or DNA amplification, which allows for ultra-fast identification of bacterial pathogens., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A theoretical study of the binding to DNA of netropsin and a bisquaternary ammonium heterocycle, SN 18071, is undertaken with an energy minimizing program based on empirical potential functions. The positioning of the ligand is achieved by force and torque calculations and its internal flexibility is taken into account. The binding preference of both drugs studied for the AT minor groove of B-DNA is shown to depend on both the electrostatic potential generated by the base sequence and the quality of the steric fit of the ligand in the groove. Ligand-DNA hydrogen bonds are shown to aid binding, but not to be essential in establishing binding preferences.

The titration viscometric investigation of the multi-mode interaction of netropsin (Nt) with (A.T) clusters of NaDNA12 and NH4DNA10 has been extended to different temperatures. The position of two boundaries on the r-scale (r= [Nt]bound/[DNA-P]) with increasing temperature steadily (rI/II) or more abruptly (rO/I) shifts to lower values. For the most (A.T) rich Nt-binding sites of modes (O), (I) and (II) this observation suggests the existence of an equilibrium between different DNA secondary structures with a different translation per base pair. The mode specific changes delta L1Nt of DNA contour length as induced by one Nt molecule proved to be almost independent of temperature. Concomitant stiffening effects increase with decreasing temperature, contrary to initial expectation. Conformational variability of (A.T) clusters may represent an essential feature in specific or selective DNA-protein interaction.

Several antibiotics, netropsin, distamycin A, actinomycin D, Hoechst 33258 and olivomycin, which demonstrate base specificity in their DNA binding properties have been found to alter the electrophoretic mobility of DNA restriction fragments in native polyacrylamide gels. The antibiotics mostly reduced the migration of larger DNA fragments, but netropsin and Hoechst 33258 were observed to increase the migration rate of several DNA fragments of intermediate size. DNA fragments of similar molecular weight which comigrate as a single gel band can at times be separated as the result of differential mobility shifts promoted by antibiotic DNA complex formations.

Oriented DNA films prepared by the wet-spinning technique have been complexed with several ligands: the anthracycline antibiotic violamycin BI, the dipeptide L-carnosine, and the oligopeptide antibiotic netropsin. The formation of the DNA-ligand complexes is accompanied by dramatic changes of the conformational flexibility of DNA. The B-A transition which occurs usually between 80% and 70% relative humidity (RH) is more or less suppressed by the ligands. Violamycin BI at a total ligand per DNA base pair ratio, rt, of approximately 0.03 and L-carnosine at rt approximately 1.5 inhibit the B-A transition of approximately 18 and approximately 0.25 base pairs per ligand molecule, respectively. Netropsin at rt = 0.2 induces a very stable B-DNA even at rather low RH (23%). The total hydration of this complex is significantly higher than for a drug-free DNA film. Netropsin-DNA complexes at rt of 0.02 and 0.01 result in an inhibition of approximately 45 base pairs per drug molecule with respect to the B-A transition.

The influence of the solvent on the binding energies to DNA of six non-intercalating antibiotics - netropsin, distamycin-3, distamycin-2, SN 18071, berenil and stilbamidine - is evaluated by combining the effect of the first hydration shell with that of bulk water. The first effect is computed by a methodology based on a spherical/point dipole model of water and limited to electrostatic interaction energies. Hydration shells are obtained which are energy optimized with respect to both water-solute and water-water interactions for the complexes and for the isolated DNA oligomers and ligands. The method allows even very large complexes to be studied in reasonable computation times. The second effect is introduced via a cavity treatment. It is shown that if the vacuum interaction energies already predict correctly the preference of the ligands for the minor groove of AT sequences of B-DNA, the introduction of the solvation effect is indispensable for reproducing the order of affinity of the ligands and for bringing the values of the complexation energies into close agreement with experimental data.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.

By means of titration viscometry a number of distinct modes could be resolved for the interaction between the antibiotic netropsin and DNA species of 50, 58, and 69 mole + (A+T) below r = 0.04 netropsin molecules bound per DNA phosphate group. The number of corresponding binding sites increases with a high power of the (A+T) content. The apparent association constants are very high (greater than 10(6) M-1, some perhaps greater than 10(6) M-1) and also rather different for most of the binding sites. It is suggested that some of these interaction modes differ in the number of hydrogen bonds formed between donors of the ligand and acceptors of the binding sites. The interaction modes were characterized quantitatively by their (species-independent) changes of DNA contour length and by the percentage of local DNA stiffening.

Six polynucleotide duplexes containing polydeoxyadenylic acid, polyadenylic acid or poly-2'-deoxy-2'-fluoro-adenylic acid in one strand, and polydeoxyuridylic acid or polydeoxythymidylic acid in the other strand have been studied by circular dichroism, ionic strength dependence of melting temperatures and binding of the DNA specific antibiotic netropsin. Circular dichroism spectra of (dA)n . (dT)n and (dA)n . (dU)n indicated the presence of the B-form of DNA, while those of (dAfl)n . (dT)n and (rA)n . (dT)n (and the corresponding (dU)n hybrids) indicated the presence of the A-form. (dAfl)n . (dT)n and (dAfl)n . (dU)n bound netropsin only slightly less than the (dA)n containing duplexes, while replacement by (rA)n decreased netropsin binding to a large degree. Since netropsin requires B-DNA for binding, it is concluded that the A to B transition is facilitated in the case of fluorine substitution in the sugar moiety, while the 2'-OH group greatly limits this conformational change.

The interaction of the oligopeptide antibiotic netropsin (Nt) with (A . T) regions of DNA is characterized by a spectrum of discrete modes. This has been revealed by viscometric analysis, at 20 degrees C and 0.2 M "counterions", for NaDNA in a preceding and for NH4DNA in this paper. The increase of DNA contour length as induced by one Nt molecule was found to depend on the special mode only, while the respective stiffening is generally higher for NH4DNA. The latter property is interpreted in terms of an enhanced flexibility, relative to that of NaDNA, of the (A . T) cluster segments before complex formation. For some of the interaction modes of the DNA-Nt systems a difference in the number of corresponding binding sites has been observed. This phenomenon is understood by assuming an influence of the counterion species upon existing equilibria between different forms of the (A . T) cluster secondary structure. Not less than 5 to 10% of the total DNA are effected in this manner. Upper limits for the local differences in the axial rise per base pair are 0.04 nm and 0.02 nm.