of K+ results in a slight (6 nm) shift of the maximum at 260 nm and in appearance of a weak negative band at 278 nm in the CD spectrum (Figure 8, compare curves 1 and 3). Figure 8. CD spectrometry of the G4-DNA. CD spectra of 200 nM tetra-molecular G-quadruplexes in 50 mM Tris-Acetate, pH 7.5 (line 1) in the presence of 10 mM KCl (line 3), and in the presence of 0.1 M LiOH (line 4). CD spectra of 200 nM monomolecular G-quadruplexes (line 2). Tetra-molecular G-quadruplexes were pre- pared, as described in ‘Materials and methods’ section. Monomolecular G-quadruplexes were prepared as described in ‘Introduction’ section using 5.5 kb G-strand. Nucleic Acids Research, 2008, Vol. 36, No. 15 5057 Electrostatic force microscopy characterization of the avidine-G4 complex An eﬃcient non-contact and non-invasive way to identify polarizable objects, usually conducting materials with AFM, is electrostatic force microscopy (EFM) (23,38). In this method the phase shift between the induced and actual tip oscillations is recorded, thus providing a sensi- tive indication for the tip and sample electrostatic interac- tion, e.g. due to charge mobility or polarizability. When performing an EFM measurement, a voltage is applied to a metallized tip after raising it above the Van der Waals (VDW) interaction height ( 10 nm) while the feedback is disabled. Attractive forces between the conductive tip and the probed object in both positive and negative bias volt- ages indicate a polarization response of the charge carriers in the measured object to the electric ﬁeld. In our measure- ment scheme, attractive forces are reﬂected as dark areas in the phase image. In Figure 9 we demonstrate that the biotin–avidine-G4 complex shows clear polarizability. Figure 9A shows topography of two molecules. Cross-sections taken on one of the molecules (continuous line, Figure 9B) and on the avidin (dotted line, Figure 9C) show heights of 2.2 and 4.5 nm respectively. The phase shift image of the same scan area as in Figure 9A during scanning 30 nm above the imaging set-point (with the same oscillation amplitude) is shown in Figure 9D, F and H for +4, 0 and 4 V, respectively, with corresponding average cross-sections in E, G and I. The dark appearance of the Figure 9. EFM measurements of avidin–biotin–G4-DNA. (A) A topography image of two complexes, where the avidin is the larger and brighter end of the molecules. (B and C) Cross-sections on the DNA and on avidin fragments of the complex from A (the molecule on the left) correspondingly. (D, F and H) Phase shift images taken 30 nm above the imaging set-point (with the same oscillation amplitude) with +4, 0 and 4 V, respectively, applied to the tip. The dark appearance of molecules in D and H indicates attraction between the molecules and the biased tip for both positive and negative voltages and therefore polarizability. At 0 V there is no detected interaction between the molecules and the tip. (E, G and I) Present an average on all the cross-sections along the left molecule from A measured at +4, 0 and 4 V correspondingly. The attractive interaction is clearly observed for 4 V and no interaction is seen at 0 V.