5054 Nucleic Acids Research, 2008, Vol. 36, No. 15 from the unbound 50-biotin–poly(dG)-poly(dC) molecules; the quality of the separation obtained with the HPLC tech- nique is, however, not suﬃcient to purify the tetra- molecular complex from the tri-molecular one. The latter complex is seen as a minor band (Figure 2B) below the major band, corresponding to the tetra-molecular com- plex, on the electrophoresis gel image. The 4[biotin–poly(dG)-poly(dC)]–avidin complex is stable at high ionic strength and does not undergo any changes for weeks at 48C. Reducing the ionic strength, however, results in its fast disintegration (Figure 3). The incubation of the saturated complex, which correspond to 3 kb bands (Figure 3, lane 3), in 20 mM Tris–HCl for 2 h results in almost complete disappearance of the bands and the appearance of a new band corresponding to 0.7 kb band (Figure 3, lane 4). These results suggest that the complex dissociates into poly(dG)-poly(dC) molecules not grouped by avidin. The dissociation can be reversed by addition of NaCl; in the presence of 0.5 M (or higher) concentration of the salts the complexes reassemble and appear as 2 and 3 kb bands on the gel (Figure 3, lane 5). The HPLC-puriﬁed tetra-molecular 1.4 kb 4[biotin– poly(dG)-poly(dC)]–avidin complexes were visualized by AFM. The complexes, collected from the HPLC column Figure 3. Dissociation and re-association of 0.7 kb poly(dG)-poly(dC)– avidin complex. The 50biotin–poly(dG)-poly(dC)–avidin complex was prepared at a poly(dG)-poly(dC) to avidin (tetramer) ratio of 5, as described in ‘Materials and methods’ section (lane 2) and was further puriﬁed by HPLC as shown in Figure 2 (lane 3). The complex was passed through a Sephadex G-25 column equilibrated with 2 mM Tris–HCl (pH 7.4) and was further incubated in the column buﬀer for 2 h at 258C (lane 4). A 0.5 M NaCl (ﬁnal concentration) was added and the incubation was continued for 24 h (lane 5). Lane 1 is 100 bp DNA Ladder. (Figure 2A), were deposited on a freshly cleaved mica sur- face and measured by AFM, as described in ‘Materials and methods’ section. Figure 4 presents an AFM image of the complex. The avidin is seen in the image as a bright sphere to which four linear poly(dG)-poly(dC) molecules are attached (Figure 4A). As can be seen in Figure 4A, there are a few molecules containing three arms, corre- sponding to an avidin–3[poly(dG)-poly(dC)] complex. The presence of a minor quantity ( 20%) of this complex is consistent with our gel-electrophoresis results (Figure 2B). The contour length distributions of poly(dG)-poly(dC) molecules attached to avidin is pre- sented in Figure 4B. The average contour lengths of more than 450 analyzed DNA molecules is 323 54 nm. We have recently demonstrated (40) that poly(dG)- poly(dC) molecules are immobilized on mica in a form related to the A-form DNA duplex, in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form corresponding to the B-form DNA duplex. The estimated contour length of the poly(dG)-poly(dC) is rather close to that expected for a double-stranded A-form DNA composed of 1400 bp (364 nm) assuming a rise per base pair of 2.6 A˚ (40). Preparation and characterization of the avidin-G4 complex A complex between four 1.4 kb biotin–poly(dG)-poly(dC) molecules and avidin, prepared as described in ‘Material and methods’ section (and the caption of Figure 2), was used as the starting material for tetra-molecular G4-DNA nanostructures production. The poly(dC) strands were separated from the biotin–poly(dG) strands connected to avidin during the size-exclusion HPLC of 4[biotin– poly(dG)-poly(dC)]–avidin at high pH. At pH > 12.5 the poly(dG) and the poly(dC) strands composing poly(dG)-poly(dC) molecules are separated from one another. The size-exclusion chromatography of the complex in 0.1 M LiOH results in a separate elution of G-strands connected to the avidin and C-strands (Figure 5). The good separation quality achieved by the size-exclusion HPLC is due to the diﬀerence in molecular masses of the 4[poly(dG)]–avidin complex containing four G-strands compared to single C-strand. The ﬁrst peak eluted from the column between 12.5 and 14.5 min corre- sponds to G-strands grouped together by avidin, while the second peak eluted between 15.5 and 18 min corresponds to C-strands. The absorption spectra of fractions eluted with the ﬁrst and the second peaks ﬁt nicely with those of G- and C-nucleotides, respectively. The poly(dG) fraction collected from the HPLC column was dialyzed against 20 mM LiCl for 4 h. During the dialysis the pH of the solution was reduced from 13.0 to 8.0 and the strands underwent folding into G4-DNA structures. The dialyzed molecules were passed through a size-exclusion HPLC column, as described in ‘Materials and methods’ section. The G4-avidin complex was collected from the HPLC column and was imaged by AFM. As seen in the AFM image (Figure 6A), each molecule is composed of a linear segment corresponding to the DNA and a brighter sphere corresponding to the avidin. The average contour length of more than 400 single analyzed molecules is equal to