Ag+-Mediated Folding of Long Polyguanine Strands to Double and Quadruple Helixes.

Metal-mediated base pairing of DNA has been a topic of extensive research spanning over more than four decades. Precise positioning of a single metal ion by predetermining the DNA sequence, as well as improved conductivity offered by the ions, make these structures interesting candidates in the context of using DNA in nanotechnology. Here, we report the formation and characterization of conjugates of long (kilo bases) homoguanine DNA strands with silver ions. We demonstrate using atomic force microscopy (AFM) and scanning tunneling microscope (STM) that binding of silver ions leads to folding of homoguanine DNA strands in a "hairpin" fashion to yield double-helical, left-handed molecules composed of G-G base pairs each stabilized by a silver ion. Further folding of the DNA-silver conjugate yields linear molecules in which the two halves of the double helix are twisted one against the other in a right-handed fashion. Quantum mechanical calculations on smaller molecular models support the helical twist directions obtained by the high resolution STM analysis. These long guanine-based nanostructures bearing a chain of silver ions have not been synthesized and studied before and are likely to possess conductive properties that will make them attractive candidates for nanoelectronics.

Ultrasmall ATP-Coated Gold Nanoparticles Specifically Bind to Non-Hybridized Regions in DNA.

Here we report the synthesis of ultrasmall (2 nm in diameter) ATP-coated gold nanoparticles, ATP-NPs. ATP-NPs can be enlarged in a predictable manner by the surface-catalyzed reduction of gold ions with ascorbate, yielding uniform gold nanoparticles ranging in size from 2 to 5 nm in diameter. Using atomic force microscopy (AFM), we demonstrate that ATP-NPs can efficiently and selectively bind to a short non-hybridized 5A/5A region (composed of a 5A-nucleotide on each strand of the double helix) inserted into a circular double-stranded plasmid, Puc19. Neither small (1.4 nm in diameter) commercially available nanoparticles nor 5 nm citrate-protected ones are capable of binding to the plasmid. The unique ability to specifically target DNA regions characterized by local structural alterations of the double helix can pave the way for applications of the particles in the detection of genomic DNA regions containing mismatches and mutations that are common for cancer cells.

Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

DARPin_9-29-Targeted Gold Nanorods Selectively Suppress HER2-Positive Tumor Growth in Mice.

Near-infrared phototherapy has great therapeutic potential for cancer treatment. However, for efficient application, in vivo photothermal agents should demonstrate excellent stability in blood and targeted delivery to pathological tissue. Here, we demonstrated that stable bovine serum albumin-coated gold mini nanorods conjugated to a HER2-specific designed ankyrin repeat protein, DARPin_9-29, selectively accumulate in HER2-positive xenograft tumors in mice and lead to a strong reduction in the tumor size when being illuminated with near-infrared light. The results pave the way for the development of novel DARPin-based targeted photothermal therapy of cancer.

Backbone charge transport in double-stranded DNA.

Understanding charge transport in DNA molecules is a long-standing problem of fundamental importance across disciplines1,2. It is also of great technological interest due to DNA's ability to form versatile and complex programmable structures. Charge transport in DNA-based junctions has been reported using a wide variety of set-ups2-4, but experiments so far have yielded seemingly contradictory results that range from insulating5-8 or semiconducting9,10 to metallic-like behaviour11. As a result, the intrinsic charge transport mechanism in molecular junction set-ups is not well understood, which is mainly due to the lack of techniques to form reproducible and stable contacts with individual long DNA molecules. Here we report charge-transport measurements through single 30-nm-long double-stranded DNA (dsDNA) molecules with an experimental set-up that enables us to address individual molecules repeatedly and to measure the current-voltage characteristics from 5 K up to room temperature. Strikingly, we observed very high currents of tens of nanoamperes, which flowed through both homogeneous and non-homogeneous base-pair sequences. The currents are fairly temperature independent in the range 5-60 K and show a power-law decrease with temperature above 60 K, which is reminiscent of charge transport in organic crystals. Moreover, we show that the presence of even a single discontinuity ('nick') in both strands that compose the dsDNA leads to complete suppression of the current, which suggests that the backbones mediate the long-distance conduction in dsDNA, contrary to the common wisdom in DNA electronics2-4.

Advances in Synthesis and Measurement of Charge Transport in DNA-Based Derivatives.

Charge transport through molecular structures is interesting both scientifically and technologically. To date, DNA is the only type of polymer that transports significant currents over distances of more than a few nanometers in individual molecules. For molecular electronics, DNA derivatives are by far more promising than native DNA due to their improved charge-transport properties. Here, the synthesis of several unique DNA derivatives along with electrical characterization and theoretical models is surveyed. The derivatives include double stranded poly(G)-poly(C) DNA molecules, four stranded G4-DNA, metal-DNA hybrid molecular wires, and other DNA molecules that are modified either at the bases or at the backbone. The electrical characteristics of these nanostructures, studied experimentally by electrostatic force microscopy, conductive atomic force microscopy, and scanning tunneling microscopy and spectroscopy, are reviewed.

Long-range charge transport in single G-quadruplex DNA molecules.

DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set-ups, and transporting significant current through individual DNA-based molecules remains a considerable challenge. Here, we report reproducible charge transport in guanine-quadruplex (G4) DNA molecules adsorbed on a mica substrate. Currents ranging from tens of picoamperes to more than 100 pA were measured in the G4-DNA over distances ranging from tens of nanometres to more than 100 nm. Our experimental results, combined with theoretical modelling, suggest that transport occurs via a thermally activated long-range hopping between multi-tetrad segments of DNA. These results could re-ignite interest in DNA-based wires and devices, and in the use of such systems in the development of programmable circuits.

Comparative electrostatic force microscopy of tetra- and intra-molecular G4-DNA.

Two forms of G4-DNA, with parallel and pairwise anti-parallel strands, are studied using atomic force microscopy. The directionality of the strands affects the molecules' structural properties (different height and length) and their electrical polarizability. Parallel G4-DNA is twice as polarizable as anti-parallel G4-DNA, suggesting it is a better electrical wire for bio-nanoelectronics.

Mono and trimethine cyanines Cyan 40 and Cyan 2 as probes for highly selective fluorescent detection of non-canonical DNA structures.

Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

High efficiency of a sequential recombinase-mediated cassette exchange reaction in Escherichia coli.

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.

Long-lived fluorescence of homopolymeric guanine-cytosine DNA duplexes.

The fluorescence spectrum of the homopolymeric double helix poly(dG) x poly(dC) is dominated by emission decaying on the nanosecond time-scale, as previously reported for the alternating homologue poly(dGdC) x poly(dGdC). Thus, energy trapping over long periods of time is a common feature of GC duplexes which contrast with AT duplexes. The impact of such behaviour on DNA photodamage needs to be evaluated.

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase.

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.

Molecular analysis of recombinase-mediated cassette exchange reactions catalyzed by integrase of coliphage HK022.

The integrase (Int) protein of coliphage HK022 can catalyze in Escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. Atomic force microscopy images have revealed that in the protein-DNA complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. This observation, together with the elucidation of intermediate co-integrates between the two circular plasmids, suggest that a sequential mechanism of the RMCE reaction is possible.

High-resolution STM imaging of novel single G4-DNA molecules.

The molecular morphology of long G4-DNA wires made by a novel synthetic method was, for the first time, characterized by high-resolution scanning tunneling microscopy (STM). The STM images reveal a periodic structure seen as repeating "bulbs" along the molecules. These bulbs reflect the helix morphology of the wires. The STM measurements were supported by a statistical morphology analysis of the DNA pitch length and apparent height relative to the surface. In the absence of X-ray and NMR data for these wires, the STM measurements provide a unique alternative to characterize the helix morphology.

Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

Redox-dependent change of nucleotide affinity to the active site of the mammalian complex I.

A very potent and specific inhibitor of mitochondrial NADH:ubiquinone oxidoreductase (complex I), a derivative of NADH (NADH-OH) has recently been discovered (Kotlyar, A. B., Karliner, J. S., and Cecchini, G. (2005) FEBS Lett. 579, 4861-4866). Here we present a quantitative analysis of the interaction of NADH-OH and other nucleotides with oxidized and reduced complex I in tightly coupled submitochondrial particles. Both the rate of the NADH-OH binding and its affinity to complex I are strongly decreased in the presence of succinate. The effect of succinate is completely reversed by rotenone, antimycin A, and uncoupler. The relative affinity of ADP-ribose, a competitive inhibitor of NADH oxidation, is also shown to be significantly affected by enzyme reduction (KD of 30 and 500 microM for oxidized and the succinate-reduced enzyme, respectively). Binding of NADH-OH is shown to abolish the succinate-supported superoxide generation by complex I. Gradual inhibition of the rotenone-sensitive uncoupled NADH oxidase and the reverse electron transfer activities by NADH-OH yield the same final titration point (approximately 0.1 nmol/mg of protein). The titration of NADH oxidase appears as a straight line, whereas the titration of the reverse reaction appears as a convex curve. Possible models to explain the different titration patterns for the forward and reverse reactions are briefly discussed.

Polarizability of G4-DNA observed by electrostatic force microscopy measurements.

G4-DNA, a quadruple helical motif of stacked guanine tetrads, is stiffer and more resistant to surface forces than double-stranded DNA (dsDNA), yet it enables self-assembly. Therefore, it is more likely to enable charge transport upon deposition on hard supports. We report clear evidence of polarizability of long G4-DNA molecules measured by electrostatic force microscopy, while coadsorbed dsDNA molecules on mica are electrically silent. This is another sign that G4-DNA is potentially better than dsDNA as a conducting molecular wire.

Chirality of silver nanoparticles synthesized on DNA.

Silver nanocrystals grown on a poly(dG)-poly(dC) double stranded DNA scaffold displayed circular dichroism at their surface plasmon excitation band. This chiral plasmon signature was not observed in a control experiment where silver nanocrystals of similar size were produced without the DNA template and adsorbed to the DNA. It is concluded that the DNA templated Ag nanocrystals had a preferred structural handedness.

High-resolution STM imaging of novel poly(G)-poly(C) DNA molecules.

High-resolution scanning tunneling microscopy (STM) imaging of single double-stranded poly(G)-poly(C) DNA molecules, made by a novel synthesis method, shows the molecules morphology. The STM images reveal a periodic structure of approximately 4 nm, seen as repeating "bulbs" along the molecules. These "bulbs" are associated with the molecule helix (the major grooves). "Nicks", two per 100 nm on the average, are observed along the DNA as well. The STM measurements were supported by a morphological statistics of the DNA molecule groove length and apparent height relative to the surface.