regions, some promoter regions, minisatellites and ribo- somal DNA. Besides their physiological relevance, gua- nine tetrads have drawn attention in the nanotechnology ﬁeld, since they were proposed as building blocks of mole- cular nanowires for nanoelectronics (18). G4-wires com- prise a large number (hundreds) of stacked guanine tetrads, thus providing better conditions for p overlap compared to the base-pairs of the canonical double- stranded DNA and higher structural rigidity and stability under various conditions (e.g. boiling, DNase). A high guanine content, having the lowest ionization potential among the DNA bases, increases the probability of charge migration through G4-wires (19–21). These proper- ties make G4-wires attractive candidates for nanoelectron- ics applications. So far, the structures made of short G-oligonucleotide sequences resulted in non-uniform polymers having gaps (non-covalently bonded backbone) between oligonucleotide fragments along the formed wires (6,8,22). These drawbacks limit the mechanical stability and stacking in so-produced wires and strongly reduce their ability to conduct current, therefore limiting their application in nanoelectronics. We have recently reported the synthesis of novel con- tinuous mono-molecular G quadruplex DNA nanostruc- tures composed of single self-folded poly(G) strands containing hundreds of stacked tetrads (18). These nano- structures are characterized by a narrow length distribu- tion and contain no gaps in their backbone. We have also demonstrated that these mono-molecular wires are char- acterized by higher charge polarizability, as compared to double-stranded DNA (23). Mono-molecular quadru- plexes, made of single intra-molecular folded G-strands, are the only long continuous G4-DNA nanostructures reported so far. Long G-quadruplexes composed of four long parallel G-strands might possess improved electric properties with respect to mono-molecular G-wires and better ability to insert interruptions in speciﬁc positions along the molecule. However, uniform inter-molecular G4-wires do not form spontaneously in solution. At high concentration, which is required for the tetra-mole- cular assembly, G-strands aggregate into bundles com- prised of a large number of strands. In order to avoid the formation of aggregates and enable only the tetra- molecular G-strand folding, we developed an approach based on the avidin–biotin interaction. The basic idea behind this approach is that the avidin-tetramer brings together four G-strands, end-labeled with biotin, therefore enabling their tetra-molecular folding at low molecular concentrations. Avidin is a glycoprotein, which consists of four identical subunits, each capable of tight biotin binding (24–28). The interaction between avidin and biotin is the strongest non- covalent interaction known in biology. The complex between the protein and the ligand is stable under various experimental conditions: pH from 2 to 13, high tempera- tures and presence of organic solvents (29–32). These unique characteristics of the complex, together with com- mercial availability of biotinylated nucleic acids and pro- teins, make the biotin–avidin system a universal tool for assembling proteins and nucleic acids (25,28,33,34). Nucleic Acids Research, 2008, Vol. 36, No. 15 5051 Here we present a novel avidin–biotin-based method that enables the formation of long, parallel, tetra- molecular G4-DNA structures. The morphology of the structures was measured by AFM and their properties were characterized by HPLC, electrophoresis, CD and absorption spectroscopy. We showed that the CD spectrum and the morphology of the tetra-molecular structures diﬀer from those of the mono-molecular G4-DNA. We demonstrated, by electrostatic force microscopy (EFM), that tetra-molecular G4-DNA nanostructures show polarizability, as their intra-molecular counterpart. MATERIALS AND METHODS Materials Unless otherwise stated, the reagents were obtained from Sigma–Aldrich (USA) and were used without further puriﬁcation. The 20-deoxyribonucleoside 50-triphosphate (dGTP) was purchased from Sigma–Aldrich (USA). Klenow fragment exonuclease minus of DNA polymerase I from E.coli lacking the 30!50 exonuclease activity (Klenow exo–) was purchased from Fermentas (Lithuania). DNA samples The oligonucleotides were purchased from Alpha DNA (Montreal, Canada). In biotinylated (dG)12-oligonucleo- tide (50biotin–(dG)12) the biotin residue is linked to the terminal base at the 50-end. All the oligonucleotides were puriﬁed by HPLC using Agilent 1100 HPLC system with a photodiode array detector. The (dC)12-oligonucleotides were puriﬁed on an ion-exchange Western Analytical Products (USA) PolyWax LP column (4.6 200 mm, 5 mm, 300 A˚ ). The oligonucleotides were eluted from the column in 10% Acetonitrile with linear K-Pi (pH 7.5) gradient from 20 mM to 0.5 M for 1 h with ﬂow rate of 0.8 ml/min. The (dG)12- and 5’biotin–(dG)12-oligonucleo- tides were puriﬁed using an ion-exchange HiTrap QHP column (5 1 ml) from Amersham-Biosciences (Sweden) at pH 13. Elution was conducted in 0.1 M NaOH and 10% acetonitrile with a linear NaCl gradient from 0.5 to 1 M for 1 h at a ﬂow rate of 0.7 ml/min. Peaks were identiﬁed by their retention times obtained from the absorbance at 260 nm. Puriﬁed oligonucleotides were desalted using pre-packed Sephadex G-25 DNA-Grade columns (Amersham-Biosciences). In order to create template- primer for enzymatic synthesis the puriﬁed 50biotin– (dG)12-oligonucleotides were incubated with their complementary counterparts in 0.1 M NaOH at a molar ratio of 1 : 1 for 15 min and further dialyzed against 20 mM Tris-Acetate buﬀer, pH 7.5, for 2 h. All the oligo- nucleotides were quantiﬁed spectrophotometrically using their respective extinction coeﬃcients. The concentrations of the (dG)12 and (dC)12 were calculated using extinction coeﬃcients of 11.7 and 7.5 mM 1 cm 1 at 260 nm, respec- tively (35).