Appl. Phys. A 66, S585–S589 (1998) Applied Physics A Materials Science & Processing  Springer-Verlag 1998 Conformational fluctuations of supercoiled DNA molecules observed in real time with a scanning force microscope G. Zuccheri 1,2 , R.Th. Dame 2,3 , M. Aquila 2 , I. Muzzalupo 2 , B. Samorì 1, * 1 Dipartimento di Biochimica, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy (Fax: +39-51/354387, E-mail: samori@alma.unibo.it) 2 Dipartimento di Chimica, Università della Calabria, Arcavacata di Rende, 87030 Cosenza, Italy 3 Department of Microbial Physiology, Free University of Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands Received: 25 July 1997/Accepted: 1 October 1997 Abstract. A scanning force microscope operating in a fluid was used to image the dynamics of supercoiled circular DNA molecules adsorbed on mica both under water and buffer. The time scales of the conformational equilibrations of molecules adsorbed on mica are controlled by frictional forces: these are modulated by the strength of adsorption that opposes the molecular mobility. We employ a method that permits the modulation of the adhesion, even to the point of completely immobilizing the molecules on the substrate and afterwards remobilizing them at will, so that their conformational fluctu- ations can start again. This method was applied to the imaging of the conformational rearrangements that take place in native supercoiled DNA molecules. The motions that were captured in the images could be interpreted in terms of supercoiling tensions that fluctuate locally. The capability of the scanning force microscope (SFM) to im- age and follow in real time the binding of protein to DNA [1] and nuclease digestion of DNA [2] has been demonstrated. These results have opened up the possibility of using the SFM to follow conformational rearrangements in a single DNA molecule and to monitor the motions of its chains in real time. The possibility of following conformational rearrange- ments in a single DNA molecule could enable us to iden- tify poorly populated states and local conformational fluctu- ations playing important roles in biological processes such as those that involve recognition and/or long-range interac- tions. Among these fluctuations could be the proposed but never demonstrated ‘bending waves’ propagating along DNA and providing site recognition information during the one- dimensional diffusion of proteins along the DNA chain [3]. In this paper we show that the conformational fluctuations taking place in native supercoiled DNA molecules on mica can be followed both in water and in buffer and that their time scale can be modulated even to the point of completely im- mobilizing the molecules on the substrate and afterwards re- mobilizing them at will so that their conformational changes * Author to whom correspondence should be addressed can start again. Very recently, Lyubchenko et al. [4] applied their method of DNA deposition on mica treated with amino- propyltriethoxy silane to the study of the shape and dynamics of supercoiled DNA molecules. While being very effective in showing the shapes of loosely and tightly supercoiled plas- mid molecules, this method produces such a strong binding of DNA to the surface that most of the chain motions are halted. Our contribution follows the same direction as the paper by Thomson et al. [5] in which the adhesion is modu- lated by changing the ionic environment on untreated mica. In our method, the change of environment is principally realized through a change in the ionic strength of the medium. 1 Experimental method Fluid SFM imaging was performed on a NanoScope III SFM (Digital Instruments, S. Barbara, CA, USA) equipped with a multimode head. A commercial tapping-mode fluid cell (Digital Instruments) was employed with electron-beam- deposited SFM probes built according to the method reported by Keller [6] on silicon nitride triangular cantilevers with a nominal force constant of 0.38 N/m. pBR322 plasmid molecules were deposited on a disc of freshly cleaved ruby mica (Mica New York Corp., NY, USA) for approximately 1 min, from a 1 μ g/ml DNA solution that contained 4 mM HEPES, 1 mM MgCl 2 at pH 6.8 - 7.4. After the deposition time, various buffers or MilliQ deionized water (Millipore, USA) were injected into the assembled fluid cell, according to the experiments reported below. SFM imaging was performed in tapping-mode at an oscil- lation frequency slightly lower than the maximum of a low- frequency oscillation peak (lower than 10 kHz) [7]. The linear scanning speed was 5–10 μ m/s for a sampling density of 15–30 nm 2 /pixel. 2 Results The application of the tapping-mode imaging to fluid SFM applications [8, 9] has permitted the observation of soft speci-