5052 Nucleic Acids Research, 2008, Vol. 36, No. 15 DNA polymerase assays The standard reaction contained 60 mM KPi, pH 7.4, 5 mM MgCl2, 5 mM DTT and 1.5 mM of dGTP and 1.5 mM dCTP, 0.2 mM Klenow exo and 0.2 mM tem- plate-primer, 50biotin–(dG)12-(dC)12. The reaction was triggered by the addition of the enzyme. The incubation was performed at 378C for time intervals indicated in the ﬁgure legends. The reaction was terminated by the addition of EDTA to a ﬁnal concentration of 20 mM. Reaction products were analyzed by size exclusion HPLC and by an agarose gel electrophoresis. HPLC separation of the polymerase products 50biotin–poly(dG)-poly(dC) produced during the enzy- matic synthesis (see above) were separated from nucleo- tides and other reaction components of the synthesis using size-exclusion HPLC. The separation was achieved with a TSK-gel G-DNA PW HPLC column (7.8 300 mm) from TosoHaas (Japan) by isocratic elution with 20 mM Tris–Acetate at pH 8.5 for 30 min at a ﬂow rate of 0.5 ml/min. Injection volumes were 20–170 ml. The separa- tion was conducted with an Agilent 1100 HPLC system with a photodiode array detector. Peaks were identiﬁed by their retention times obtained from the absorbance at 260 nm. Preparation of avidin–Poly(dG)-Poly(dC) complex Egg-white avidin (0.2 mM) was incubated with 50biotin– poly(dG)-poly(dC) (1 mM in molecules) in 0.2 M CAPS buﬀer (pH 10.5) and 0.5 M NaCl, for 4 h at 258C. The concentration of avidin was calculated using extinc- tion coeﬃcient of 97.4 mM 1 cm 1 at 280 nm (36). The avidin–50biotin–poly(dG)-poly(dC) complex was puriﬁed from unbound 50biotin–poly(dG)-poly(dC) molecules using size-exclusion HPLC. The separation was achieved with a TSK-gel G-DNA PW HPLC column (7.8 300 mm) from TosoHaas (Japan) by isocratic elution with 20 mM Tris–Acetate (pH 8.5), 0.5 M NaCl, for 30 min at a ﬂow rate of 0.5 ml/min. Peaks were identi- ﬁed by their retention times obtained from the absorbance at 260 nm. The purity of the avidin–50biotin–poly(dG)- poly(dC) complex was conﬁrmed by agarose gel electrophoresis. Preparation of avidin–G4-DNA complex The avidin–50biotin–poly(dG)-poly(dC) complex obtained as described above was pretreated for 20 min at room temperature in 0.1 M LiOH in order to dissociate poly(dG)-strands connected to avidin from poly(dC)- strands. The separation was carried out with a size- exclusion TSK-gel G-DNA PW HPLC column (7.8 300 mm) from TosoHaas (Japan) by isocratic elution with 0.1 M LiOH for 30 min at a ﬂow rate of 0.5 ml/min. Peaks were identiﬁed by their retention times obtained from the absorbance at 260 nm. The avidin– poly(dG) fraction was collected and dialyzed against 20 mM LiCl for 4 h. During the dialysis the pH of the solution was reduced and the G-strands folded. The dia- lyzed complex was chromatographed on TSK-gel G-DNA PW HPLC column (7.8 300 mm) from TosoHaas (Japan) in 0.1 M Tris–Acetate (pH 7.5). The main fraction was collected; the late fraction enriched with tri-molecular avidin–50biotin–poly(dG) complex was discarded. CD and absorption spectroscopy CD spectra were recorded at 258C with an Aviv Model 202 series (Aviv Instrument Inc., USA) Circular Dichroism Spectrometer. Each spectrum was recorded from 220 to 340 nm and was an average of 5 scans. Absorption spectra of the synthesized products were recorded with a Jasco V-630 spectrophotometer (Japan). AFM imaging Atomic force microscopy was performed on the molecules adsorbed onto mica surfaces. A 20 ml of 1.0–2.0 nM (in molecules) Avidin–DNA and G4-DNA samples in 50 mM HEPES or Tris–Acetate, pH 7.5, containing 0.5–2.0 mM MgCl2 were incubated on a freshly cleaved moskovite mica plates for 5 min, washed with distilled water and dried with nitrogen gas. AFM images were obtained with two systems: (i) a Solver PRO (NT-MDT, Russia) AFM in a non-contact (tapping) mode using 130 mm long Si-gold-coated cantilevers (NT-MDT, Russia) with resonance frequency of 119–180 kHz. (ii) Images and electrostatic polarizability were measured with Nanotec Electronica S.L. Madrid, in dynamic mode. Soft cantilevers (OMCL-RC800PSA, Olympus Optical Co., Ltd) of nominal force constant 0.3 Nm 1, resonance frequency 75–80 kHz and tip radius 15–20 nm were used (the tip radius was measured with a scanning electron microscope). The cantilever was oscillated at its resonance frequency while the amplitude and the phase, relative to the oscillatory driving force, were measured through the tip deﬂection signal as detected by the photo-detector. The feedback was performed on the amplitude signal channel. The images were ‘ﬂattened’ (each line of the image was ﬁtted to a 2nd-order polyno- mial, and the polynomial was then subtracted from the image line) by the AFM’s image processing software. The images were analyzed and visualized using a Nanotec Electronica S.L (Madrid) WSxM imaging soft- ware (37). Electrostatic force microscopy (EFM) polarizability measurements The electrostatic polarizability of the molecules was checked by EFM using various methods described in details in (23,38). The EFM results presented here were measured in retrace mode with a tip lift of 30 nm above the topography set point height (and above the eﬀective VDW force range, where the electrical interaction is dominant) at 0, 4 and +4 V applied to the tip. The phase shift signal that is compared for the diﬀerent molecules at the various voltages is integrated from the cross-sections along the molecule. For electrical functionality all the can- tilevers used in this work were sputter-coated with AuPd at a nominal thickness of 15–30 nm. As a result the reso- nance frequency was reduced to 60 kHz. While scanning, the cantilevers were oscillated at low amplitude of up to