Nucleic Acids Research, 2008, Vol. 36, No. 15 5055 Figure 4. AFM image of avidin–50biotin–poly(dG)-poly(dC). (A) AFM image of 1.4 kb 50biotin–poly(dG)-poly(dC)–avidin complex. The complex was formed at a biotin–poly(dG)-poly(dC) to avidin ratio of 5, as described in ‘Materials and methods’ section, and was further puriﬁed by HPLC as shown in Figure 2. Molecules were deposited on mica and measured, as described in ‘Materials and methods’ section. The avidin is seen as a brighter spot in the middle of the complex. (B) Statistical contour length analysis of more than 400-well separated DNA molecules attached to avidin. Inset: Enlargement of a 50biotin–poly(dG)-poly(dC)–avidin complex. Figure 5. Size-exclusion HPLC separation of four-molecular 1.4 kb 50-biotin–poly(dG)–avidin complex from poly(dC) in base. The 50biotin–poly(dG)-poly(dC)–avidin complex was prepared at a poly(dG)-poly(dC) to avidin ratio of 5, as described in ‘Materials and methods’ section, and was further puriﬁed by HPLC as shown in Figure 2. The complex was pretreated for 20 min at room temperature in 0.1 M LiOH in order to dissociate poly(dC)-strands from poly(dG)- strands connected to avidin. The separation was achieved with a size- exclusion G-DNA-PW HPLC column (7.8 300 mm) by isocratic elution with 0.1 M LiOH for 30 min at a ﬂow rate of 0.5 ml/min. 275 25 nm (Figure 6C). Statistical AFM morphology imaging analysis shows that the contour length of the G4-DNA molecules is somewhat shorter, although within the experimental error, to the length of the initial poly(dG)-poly(dC) (323 54 nm, see Figure 4) suggesting a tetra-molecular mechanism of the G-strands assembling. The morphology of the tetra-molecular G-quadruplexes reported here was compared with the morphology of mono-molecular G4-DNA reported by us earlier (18). The latter mono-molecular quadruplexes are prepared by folding a single G-strand four times as described in (18). For the sake of comparison, mono- molecular G-quadruplexes were made from the same amount of G-bases ( 5.5 kb) as the tetra-molecular ones (4 1.4 kb). Note that in mono-molecular G-quadruplexes all G-bases belong to the same chain, while in tetra-molecular G-quadruplexes they are equally distributed between the four chains composing the DNA. Moreover, in the 4-folded molecules every two strands run in opposite directions, while in the latter molecules all the four strands run in the same 30–50 direction. Both types of molecules were deposited on a freshly cleaved mica surface and measured by AFM. Figure 6A and B shows images and C and D comparative contour length analyses of the tetra- and mono-molecular G4-quadruplexes, correspondingly. The statistical analy- sis (Figure 6C and D) shows that the contour length of the tetra-molecular quadruplexes (275 25 nm) is shorter than that of the mono-molecular ones (351 44 nm). The average height of the tetra-molecular quadruplexes (2.2 0.2 nm) is noticeably larger than that of the mono-molecular quadruplexes (1.0 0.2 nm). The appar- ent heights were extracted from cross-sections of hundreds of molecules of each type measured under identical conditions. Figure 7 presents height proﬁles taken along the marked lines of two typical tetra-molecular single G4-wires. The height of the avidin is approximately equal to 5 nm, while the height of the G4-DNA connected to it is approximately equal to 2.5 nm. It is clearly evident from Figure 7 that the height of the DNA in the complex is not inﬂuenced by the protein; the same height was measured close (2.5 nm) to and opposite to the site of the avidin’s attachment. CD spectra of tetra- and mono-molecular G-quadruplexes are shown in Figure 8. As seen in the ﬁgure, the CD spectrum of the mono-molecular quadru- plexes is characterized by a positive band at 256 nm and a negative band at 276 nm, while the spectrum of tetra- molecular quadruplexes has only a positive band at