5050–5060 Nucleic Acids Research, 2008, Vol. 36, No. 15 doi:10.1093/nar/gkn459 Published online 28 July 2008 Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin–biotin recognition Natalia Borovok 1 , Natalie Iram1, Dragoslav Zikich 1 , Jamal Ghabboun 2 , Gideon I. Livshits2, Danny Porath2,3 and Alexander B. Kotlyar1,4,* 1Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978, 2Physical Chemistry Department, 3Center for Nanoscience and Nanotechnology, The Hebrew University, Jerusalem 91904, Israel and 4The Center for Nanoscience and Nanotechnology, Tel Aviv University Received May 4, 2008; Revised July 1, 2008; Accepted July 2, 2008 ABSTRACT 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 struc- tures 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 con- nected 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 assem- bly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA mole- cules 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. INTRODUCTION G-rich DNA sequences containing runs of guanines (dG) can form G-quadruplex structures. These structures, com- monly named G4-DNA, are comprised of stacked tetrads; each of the tetrads arises from the planar association of four guanines by Hoogsteen hydrogen bonding. The potential biological role of G4-DNA stimulated investiga- tion of the physico-chemical properties of these structures. Most of the studies have been performed using short (16–32 bases) G-rich telomeric oligonucleotides that are stable only in the presence of stabilizing cations (K+ or Na+) (1–5); removal of the cations results in a complete unfolding of the G-quadruplexes. Short G-rich oligonu- cleotides were shown to assemble spontaneously into long molecular wires in the presence of proper monovalent cations (6–8). These wires comprise a large number of G-oligonucleotide fragments assembled in a tetra-helix fashion and not covalently connected to one another. G-quadruplex structures are very polymorphic and are characterized by various molecularity, topology and strand orientation (9). G-quadruplex DNA may be formed from one, two and four separate DNA strands, thus termed mono-, bi- and tetra-molecular G4-DNA, correspondingly (10–15). In the tetra-molecular structures, each one of the four strands contributes one G-residue to form a tetrad. In the bi-molecular structures, each strand contributes two gua- nines to each tetrad and in the mono-molecular structures all the G-residues are coming from the same strand. G4-DNA forming sequences were found at the ends of chromosomes in the so-called telomeric regions, as well as in transcriptional regulatory regions in several onco- genes (16,17), immunoglobulin heavy chain switch *To whom correspondence should be addressed. Tel: 972 3 6407138; Fax: 972 3 6406834; Email: s2shak@post.tau.ac.il Correspondence may also be addressed to Danny Porath. Tel: 972 2 6586948; Fax: 972 2 6586987; Email: porath@chem.ch.huji.ac.il 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.