Natalia Borovok

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.

In vitro synthesis of uniform poly(dG)–poly(dC) by Klenow exo− fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo− fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.

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.

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60‐120 MW/cm2) of an oxygen‐free aqueous solution of pyranine (8‐hydroxypyrene‐l,3,6‐tri‐sulfonate) by the third harmonic frequency of an Nd‐Yag laser (355 nm) drives a two successive‐photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ+). The oxidized product, φ+, is an intensely colored compound (Λmax= 445 nm, ε= 43 000 ± 1000 M−1 cm−1) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ+ is stable, having a lifetime of ‐10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H+. In alkaline solution the favored electron acceptor is the ground‐state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M‐l cm−1. The oxidized radical can be reduced either by φ‐ or by other electron donors. The apparent second‐order rate constants of these reactions, which vary from 106 up to 109M−1 s−1, are slower than the rates of diffusion‐controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3‐D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His‐N or Cys‐S) and the observed rate constants of Cu(II) reduction.

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions.

Three types of DNA: ∼2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)–poly(dC)], ∼2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)–poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)–poly(dC) is ∼30% shorter than that of poly(dA)–poly(dT) and the plasmid. This led us to suggest that individual poly(dG)–poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A‐form of DNA in contrast to poly(dA)–poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B‐form.

Dynamics of hippocampal protein expression during long-term spatial memory formation

Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein trafficking, enhancement of metabolic activity, and Wnt signaling pathway during the steep phase of memory formation; and (3) cytoskeleton organization proteins. Taken together, this study clearly demonstrates dynamic assembly and disassembly of protein-protein interaction networks depending on the stage of memory formation engrams.

Synthesis and Assembly of Conjugates Bearing Specific Numbers of DNA Strands per Gold Nanoparticle

Here, we present a relatively simple, efficient, and high-yielding polymerase-based method for the synthesis of 15 nm gold nanoparticle conjugates bearing a specific number of 25 base oligonucleotide strands. We have shown that the conjugates bearing one or two oligonucleotide strands per particle, with the conjugates comprising a single complementary strand, self-assemble into nanoparticle dimers and trimers, respectively. Incubation of fully coated AuNPs, containing tens of oligonucleotide strands, with a conjugate bearing a single complementary strand leads to the formation of flower-shaped structures. The assembly of particles into nanoparticle structures shown here is a prerequisite for more complex controlled assembly of particles into three-dimensional macrostructures.

Synthesis of novel poly(dG)–poly(dG)–poly(dC) triplex structure by Klenow exo− fragment of DNA polymerase I

The extension of the G-strand of long (700 bp) poly(dG)–poly(dC) by the Klenow exo− fragment of DNA polymerase I yields a complete triplex structure of the H-DNA type. High-performance liquid chromatography analysis demonstrates that the length of the G-strand is doubled during the polymerase synthesis. Fluorescence resonance energy transfer analysis shows that the 5′ ends of the G- and the C-strands, labeled with fluorescein and TAMRA, respectively, are positioned close to each other in the product of the synthesis. Atomic force microscopy morphology imaging shows that the synthesized structures lack single-stranded fragments and have approximately the same length as the parent 700 bp poly(dG)–poly(dC). CD spectrum of the polymer has a large negative peak at 278 nm, which is characteristic of the poly(dG)–poly(dG)–poly(dC) triplex. The polymer is resistant to DNase and interacts much more weakly with ethidium bromide as compared with the double-stranded DNA.

Redox photochemistry of thiouredopyrenetrisulfonate.

Abstract 1-Thiouredopyrene-3,6,8-trisulfonate (TUPS) has recently been used as a photoinduced covalent redox label capable of reducing various cofactors of proteins. A new reaction of this dye, whereby its excited triplet state oxidizes suitable electron donors, is now reported. The characteristic difference spectrum of the reduced radical of TUPS is determined. We also observe the self-exchange electron transfer between two TUPS molecules in their triplet excited states and determine the reaction scheme and the rate constants of the various pathways in the process of triplet depletion. The ability of photoexcited TUPS to withdraw an electron from reduced cytochrome-c is also observed. It is thus demonstrated that TUPS is an appropriate photoinduced covalent redox label for initiating both the oxidative and reductive phases of electron transfer processes in biological macromolecules.