DNA-Based Molecular Nanowires

We developed a novel method for enzymatic synthesis of µm-long uniform and continuous polyG-polyC double stranded molecules [1,2,3]. The enzymatic mechanism for the synthesis was released and made publicly available [1]. We remark that this is a central success of the whole project: the attainment of this synthesis protocol allows us now to have DNA-based molecules of different helical motifs (double, triple and quadruple helix, see below) of controllable quality and of length ranging between hundred nm and µm. Such lengths are necessary for the envisaged technological exploitation of the molecules. On the contrary, very short segments on which almost all the single-molecule measurements on DNA are done would not allow the successive steps towards devices and self-assembling circuits. We have demonstrated that the extension of the G-strand of 700 base pairs poly(dG)-poly(dC) by Klenow exo fragment in the presence of dGTP yields a complete poly(dG-dG)-poly(dC) triplex [4]. HPLC analysis of the synthesis products shows that the amount of dG-bases incorporated into the dG-strand during the synthesis is equal to the amount of dG-bases in poly(dG)-poly(dC); the length of the poly(dG)-strand is thus doubled during the synthesis. Direct AFM imaging of the molecular morphology shows that the poly(dG)-poly(dC) and the poly(dG-dG)-poly(dC) have almost the same length, and that no single-stranded fragments are present in the synthesized product. These data strongly indicate that the de novo G-strand is associated with the poly(dG)-poly(dC) duplex. The doubling of the poly(dG) strand was monitored by FRET analysis. [1] A.B. Kotlyar, N. Borovok, T. Molotsky, L. Fadeev, M. Gozin, "In vitro synthesis of uniform poly(dG)- poly(dC) by Klenow exo- fragment of polymerase I", Nucl. Acid Res. 33, 525 (2005). [2] E. Shapir, H. Cohen, N. Borovok, A.B. Kotlyar, D. Porath, "High-resolution STM imaging of novel poly(G)-poly(C) DNA molecules", J. Phys. Chem. B 110, 4430-4433 (2006). [3] A.B. Kotlyar, N. Borovok, T. Molotsky, H. Cohen, E. Shapir, D. Porath, "Long Monomolecular Guanine-Based Nanowires", Adv. Mater. 17, 1901 (2005) (with journal cover - cover of the year). [4] A.B. Kotlyar, N. Borovok, T. Molotsky, D. Klinov, B. Dwir, E. Kapon, "Synthesis of novel poly(dG-poly(dG)-poly(dC) triplex structure by Klenow exo- fragment of DNA polymerase I", Nucl. Acid Res. 33, 6515-6521 (2005).

The above molecules were used as the starting point for designing new procedures for complexation with metal ions and production of µm-long monomolecular G4-DNA nanowires [1]. Synthesis of the above DNA-derivatives was optimised and complemented by characterization using standard (bio) chemical methods [1], Atomic and Electrostatic Force Microscopy (AFM and EFM) [1,2], Scanning Tunnelling Microscopy (STM) [3,4] and Spectroscopy (STS) [5], electrical transport measurements, and theory [6]. The efficiency of the method for the synthesis of G4-DNA was optimised. G4-DNA was originally synthesized from long polyG-polyC double-stranded DNA that is produced enzymatically with Klenow exonuclease minus DNA polymerase. The strands were separated chromatographically at high pH, and the G4-DNA molecules were formed upon lowering of the pH. It was found that dCTP (cytosine tri-phosphate) can be replaced during the enzymatic synthesis with an oligomer of cytosine (typically 20-mer), to obtain a double helix of polyG with unconnected oligomers of C. This improved dramatically the separation yield of the polyG from the fragmented oligomers of C under basic conditions, and hence enabled us to obtain much higher yields in G4-DNA production, although less size uniformity on the surface is observed [7] [1] A.B. Kotlyar, N. Borovok, T. Molotsky, H. Cohen, E. Shapir, D. Porath, "Long Monomolecular Guanine-Based Nanowires", Adv. Mater. 17, 1901 (2005) (with journal cover - cover of the year). [2] Hezy Cohen, Tomer Sapir, Natalia Borovok, Tatiana Molotsky, Rosa Di Felice, Alexander B. Kotlyar and Danny Porath, "Polarizability of G4-DNA Observed by EFM Measurements" - Submitted. [3] E. Shapir, H. Cohen, N. Borovok, A.B. Kotlyar, D. Porath, High-resolution STM imaging of novel poly(G)-poly(C) DNA molecules , J. Phys. Chem. B 110, 4430-4433 (2006). [4] E. Shapir, J. Yi, H. Cohen, A. Kotlyar, G. Cuniberti, and D. Porath, "The puzzle of contrast inversion in DNA STM imaging", J. Phys. Chem. B Lett. 109, 14270 (2005). [5] E. Shapir, A. Calzolari, C. Cavazzoni, D. Ryndyk, G. Cuniberti, R. Di Felice, and D. Porath, "Electronic structure of single DNA molecules resolved by transverse scanning tunneling spectroscopy" - Submitted. [6] R. Di Felice, A. Calzolari, A. Garbesi, S.S. Alexandre, J.M. Soler, "Strain-dependence of the electronic properties in periodic quadruple helical G4-wires", J Phys. Chem. B 109, 22301 (2005); A. Calzolari, R. Di Felice, E. Molinari, A. Garbesi, J. Phys. Chem. B 108, 2509 (2004); J. Phys. Chem. B 108, 13058 (2004); M. Cavallari, A. Calzolari, A. Garbesi, and R. Di Felice, "Stability and Migration of Metal Ions in G4-wires by Molecular Dynamics Simulations", J. Phys. Chem. B 110, 26637 (2006). [7] N. Borovok et. al., "Enzymatic Synthesis of long uniform poly(dG) strands and their folding into G4-DNA nanostructures" - Submitted.

The electronic structure of periodic G4-wires indicates that a pure band-like conduction mechanism is unlikely. However, the p-p overlap between consecutive planes originates channels for helectron/hole motion [Arrigo Calzolari, Rosa Di Felice, Elisa Molinari, Anna Garbesi, "G-Quartet Biomolecular Nanowires", Appl. Phys. Lett. 80, 3331 (2002); A. Calzolari, R. Di Felice, E. Molinari, A. Garbesi, J. Phys. Chem. B 108, 2509 (2004); J. Phys. Chem. B 108, 13058 (2004)]. The electronic structure parameters (band gaps and widths) are very sensitive to various geometrical factors, such as twisting and axial strain [R. Di Felice, A. Calzolari, A. Garbesi, S.S. Alexandre, J.M. Soler, "Strain-dependence of the electronic properties in periodic quadruple helical G4-wires", J Phys. Chem. B 109, 22301 (2005)]. The optical properties of nucleobases and their assemblies were computed from first principles in the framework of Time-Dependent DFT for the first time, and compared to available experimental data with success [D. Varsano, R. Di Felice, M.A.L. Marques, A. Rubio, "A TDDFT study of the excited states of DNA bases and their assemblies", J. Phys. Chem. B 110, 7129 (2006)]. The optical investigations are currently continuing with the implementation and test of circular dichroism (CD) spectra at the same level of theory: reliable CD spectra computed in this way can be employed for the interpretation of in-situ post-synthesis data, since CD is often used for routine characterization

It is found that the metal species in the inner channels affects the overall stability of the quadruple helical conformation. The shorter the quadruplexes, the more sensitive they are to the degree of coordination and they may unfold when they are not completely filled with metal cations. Instead, long quadruplexes can remain in the folded state even in the absence of inner cations or in the presence of the smallest Li ions [10. Manuela Cavallari, Arrigo Calzolari, Anna Garbesi, and Rosa Di Felice, "Motion of metal ions in G4-wires by Molecular Dynamics simulations", J. Phys. Chem. B 110, 26337 (2006).]. These simulations were undertaken to explain some aspects of the synthesis.

Direct electrical transport measurements on a complex sequence of 26 base-pair double-stranded DNA showed high currents (220 nA@2V) [1,2]. These experiments were performed when the ssDNA molecules are arranged in a mono-layer on a gold surface. Some of them hybridise to a complementary strand that is attached to gold nanoparticles at the opposite ends and no non-specific interaction along the molecule with a hard surface occurs. A conductive AFM tip is utilized to both scan the sample and contact the gold nanoparticles that are connected to the surface through the dsDNA. A comprehensive set of control experiments, including I-V curves obtained while stretching the DNA molecules, confirms these results. The results of these experiments have been recently published [1]. Further experiments utilizing this experimental method are under way to characterize sequences of varying length, sequence and of G4-DNA. Further measurements were performed on mono-layers of ssDNA, dsDNA connected with thiols only on one end to the surface and on dsDNA thiolated on both ends. These measurements were done without the gold nanoparticles, confirmed the above results and demonstrated the crucial role of the covalent attachment of the molecules to the electrodes [2]. [1] H. Cohen, C. Nogues, R. Naaman, D. Porath, "Direct measurement of electrical transport through single DNA molecules of complex sequence", Proc. Natl. Acad. Sci. USA 102, 11589 (2005). [2] Hezy Cohen, Claude Nogues, Daniela Ullien, Shirley Daube, Ron Naaman and Danny Porath, "Electrical characterization of self-assembled single- and double-stranded DNA monolayers using conductive AFM", Faraday Discussions (with volume cover) 131, 367 (2006).

As a preliminary experimental-theory collaboration before the beginning of the project, a simple fishbone model was initially formulated and applied to investigate the role of nucleobase-backbone coupling in the electrical behaviour: the evidence of an excitation gap in measured transport curves was explained [1]. Later, the model was refined to take into account the environmental effects, in particular the presence of a solution represented as a thermal bath. The results show important modifications of the quantum conductance at the Fermi level due to electron-phonon coupling, and allow for an interpretation of recent experiments [2,3]. We remark that this theoretical activity, devoted to environmental effects, was undertaken as a follow-up to reviewers' comments at the evaluations, which encouraged a tighter experiment-theory connection and concerted research efforts. [1] G. Cuniberti, L. Craco, D. Porath, C. Dekker, "Backbone-induced semi-conducting behavior in short DNA wires", Phys. Rev. B 65, R241314 (2002). [2] R. Gutierrez-Laliga, S. Mandal, and G. Cuniberti, "Quantum Transport through a DNA Wire in a Dissipative Environment", Nano Letters 5, 1093 (2005). [3] R. Gutierrez-Laliga, S. Mandal, and G. Cuniberti, "Dissipative effects in the electronic transport through DNA molecular wires", Phys. Rev. B 71, 235116 (2005).

Some relations have been established between the kinetics of incoherent hopping charge transport in bridged large scale chemical systems or in a single-component duplex DNA, and the electrical properties of these systems connected between two electrodes [(a) M. Bixon and J. Jortner, "Incoherent charge hopping and conduction in DNA and long molecular chains", Chem. Phys. 319, 273 (2005); (b) M. Bixon and J. Jortner, "Shallow traps for thermally induced hole hopping in DNA", Chem. Phys. 326, 252 (2006)]. The kinetic model can be applied for the description of incoherent charge transport in a donor-acceptor pair connected by two electrodes. In summary, different behaviours of the current are found in the low-voltage and high-voltage regimes. In both limits the current intensity depends on the details of the voltage drop through the junction and on the lead-molecule interfaces, which are still to be unravelled before a quantitative theory is disclosed for the direct simulation of experimental conditions.

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