5058 Nucleic Acids Research, 2008, Vol. 36, No. 15 molecules in the phase shift images, when 4 V are applied, as expected, no molecules are seen at zero-bias. This suggests that the molecules are good candidates for conductivity measurements and if the wires will be found conductive, they will be very useful for nanoelectronic applications, as nanoelectronic wires. DISCUSSION We described here the procedure for preparation of novel long continuous tetra-molecular G4-wires based on avidin–biotin interaction. An avidin-tetramer, that can bind up to four biotin molecules (24–28), was used to bring together four G-strands, end-labeled with biotin and enabled their tetra-molecular folding. The avidin– biotin complex is extremely stable under various experi- mental conditions (pH from 2 to 13, high temperatures, presence of organic solvents) (29–32) and is particularly useful for preparation of tetra-molecular G4-wires. The procedure of the G4-wires production consists of four main stages schematically illustrated in Figure 10: (i) The synthesis of 50-biotinylated-poly(dG)-poly(dC) molecules. The synthesis includes a 50biotin–(dG)12- (dC)12 template-primer extension in the presence of dGTP and dCTP by Klenow exo (39) (Figure 10A); (ii) Formation of a complex between the avidin and four biotinylated poly(dG)-poly(dC) molecules (Figure 10B); (iii) Separation of the poly(dC) strands from poly(dG)- strands connected to the avidin (Figure 10C); (iv) Assembling of four G-strands connected to avidin into tetra-molecular G4-DNA structures (Figure 10D). The above procedure yields G4-DNA molecules having an average length approximately equal to that of the parent poly(dG)-poly(dC) molecules (Figures 4 and 6). This result, together with the similar distance between adjacent base pairs as in dsDNA (41,42) and adjacent tetrads in G-qadruplexes (1,2,43,44), strongly suggests a tetra- molecular mechanism of G-strands assembly into G-quadruplexes. The complex between an avidin-tetramer and four poly(dG)-poly(dC) molecules is unstable at low ionic strength and undergoes dissociation at room temperature (Figure 3). The dissociation is reversible; an increase of the ionic strength leads to almost complete reassembly of the complex. The avidin-tetramer, as well as the complex, is very stable and is not disturbed by heat or manipulations like multiple washings and dilutions (28,31,32). However, attachment of long negatively charged molecules such as DNA to the avidin can destabilize the complex. At low ionic strength the electrostatic repulsion between the DNA molecules exceeds the forces that stabilize the avidin- tetramer (hydrophobic interactions and hydrogen bonds between the sub-units). Under these conditions, the disso- ciation reaction becomes energetically favorable and Figure 10. Scheme of tetra-molecular G4-DNA production. (A) The synthesis of 1.4 kbp poly(dG)-poly(dC) molecules biotin labeled at 50-end of the poly(dG)-strand. (B) Formation of the complex between avidin and four biotinylated poly(dG)-poly(dC) molecules. (C) Separation of poly(dC) strands from biotin–poly(dG)-strands connected to avidin in 0.1 M LiOH. (D) Assembly of four G-strands connected to avidin into tetra-molecular G4-DNA structures.