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Novel enzymatic synthesis of poly(G)-poly(C) and its mechanistic explanation. Synthesis and characterization of novel triplex nanostructures

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).

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