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Surface Investigation, 1998, VoL 14, pp. 247-251 Reprints available directly from the publisher Photocopying permitted by license only

© 1998 OPA (Overseas Publishers Association) N. V Published by license under the Gordon and Breach Science Publishers imprint Printed in India

Application of a Scanning Probe Microscopy to the Study of DNA Conformati on
M. O. Gallyamov, O. A. Pyshkina, V. G. Sergeev, and I. V. Yaminsky
M. V. Lomonosov Moscow State University, Center of Prospective Technologies, Moscow, Russia Received 14 June, 1997; accepted 24 September, 1997

Conformational properties of DNA have been studied by scanning probe microscopy and the advantages of this method have been shown for the study of biopolymer morphology. It has been shown that in aqueous-alcohol solutions the linear DNA has the toroid-like conformation without any compacting agents. The conformational transition of the supercoiled DNA into the unfolded circle occurs under the cyclic DNA-surfactant interaction, Morphology of linear DNA in the complex with a surfactant has been determined, It has been assumed that the toroid-like conformation of linear DNA is energetically favorable when the DNA charge is neutralized.

To study the morphology and conformational state of natural biopolymers which are in aqueous solutions or adsorbed on the ',skt"ace of different substrates, many physicalchemical methods (circulardichroism, dispersion of optical rotation, double refraction in flux, X-ray diffraction studies etc.). have been used. However, to visualize and determine the morphology of sucha complicated natural biopolymer as DNA, the application of atomic-force methods (AFM) [lj .andscanning tunneling microscopy (STM) [2] is more prospective. In the given work the complex STM and AFM investigations of the DNA morphology were carried out in different conditions and it has been shown that the obtained AFM and STM images of the objects studied are in good agreement with the results obtained earlier. Mica (muscovite) was used as a substrate in AFM investigations, high-oriented pyrographite was applied in STM investigations. A drop of the solution (2 Ill) containing the structures studied was deposited on the freshly sheared substrate surface, then the samples were held for 5 min, washed with distilled water, dried in air and investigated by probe microscopy methods. A scanning tunneling microscope SCAN-8 (Russia) [3] was used, tunnel parameters were varied in the range: It from 0.2 to 1 nA, VI from 50 to 1000 mY. The AFM investigations were carried out in the contact and interrupted contact regimes [4] by Nanoscope-3 (USA).

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F ig ure 1. Th e AFM i mage of lin e ar D NA fro m c hick e ryt hr o cyt e s de posi ted fro m th e 0 .05 mo le TB E b u ffe r with 50 % iso propanol solut ion on the m ic a surfa ce . Co ntac t reg ime . Th e frame s ize is 2.5 x 2 .5 11 m, t he g ra y sc al e i s IS n rn,


APPLI CA nON OF A SCANNIN G PROBE MI CROSCOP Y

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Figure 2. The AFM image of the cyclic DNA complex (p UC 19) with a s urfa c ta nt (ce tyl trimethylammon ium bromide ) adsorb ed o n th e mica surf ace , cont act regime . The f rame size is 0.45 x 0.45 urn, the gray scale is 15 nm .

Figure 1 illustrates the AFM image of linear DNA from chick erythrocytes depo sited fro m the TBE buffer with 50% is opropanol. It ha s been sh o wn th at in these conditions DNA has the toroid-like morpholog y eve n if the c o m pac ting agent s are absen t. We assume that if alcoh ol is pre sent (analogous result were obt ained for methanol, ethanol and isobutan ol), the appro ach of Na" counterions with negativ e ch arged DNA phosphate gro up s occurs up to the c omplete charge sc re e ni ng on a ma cr omole cule. As a r esult the intern al stres ses release the DNA double spiral, which takes the most energ eti cally advantageous sp ace co nfo rma tio n, occurs. Howev er, the type of the most advant ageou s DNA co nforma ti o n appeared to depend on the initial macromolecule conformation . Figur e 2 illu str ates the AFM image of the cyclic DNA co m plex (p UC 19) with the opp osite-charged s urfac e -ac tive su bs ta nce CTAB (c et yltrimethyl ammonium bromide). Taking into ac c ount th at the init ial cyclic DNA was in the s upe rtw is ted conformati on , one c a n c oncl ud e that , if th e neg ati ve c harg e is neutr ali zed , DNA with CTAB take s another most advantageous c o nforma tio n - an unfold ed cir cle . It should be noted that the appli c atio n of AFM allowed us to an swer the question about the morphol og y of cycli c DNA in the c o m ple x with a surfa c tant. As w as sho wn earlier [5] the compl exes of linear DNA with a surfactant also rem ain a c ompact co n forma tio n in low -polarity o rga nic media . Howev er , it is not possible to determin e the morpholo gy of DNA includ ed in th e comple x by the usu al physi c al- chem ic al method s , suc h as visco simetry , double refraction, UV spectro sc opy etc. As is seen fr o m Fig s. 3 a nd 4 the anal ogous probl em was solv e d by STM . Fi gur e 3 illu str ate s the STM im age of the c omplex of line ar DNA (fro m chi ck eryth rocyt e) with an opp os ite char ged surf ac ta nt (DSDMAC - d ist e ary ld ime th ylammo niu m ch lori de) pre p are d fro m a n aq ueo us


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Fi gu re 3. T he ST M im age o f the co mplex of li near DNA (fro m c hick eryt hrocyte) wit h a surfactant (d isteary ldimet hyi am moniu m c hlorid e ) prep ared i n aq ueo us so lutio n and adso rbed on the pyrogra ph ite s urface . Th e co nstan t c urre nt re gim e . Th e frame size is 0.4 x 0.4 urn , t be gray scale is 15 11 m .


APPLICAnON OF A SCANNING PROBE MICROSCOPY

251

Figure 4. The STM image of the complex of linear DNA (from chick erythrocyte) with a surfactant (distearyldimethylamrnonium chloride) prepared in chloroform and adsorbed on the pyrographite surface . A constant current regime. The frame size is 0 .13 x 0 .13 urn, the gray scale is 6 nm.

solution. Figure 4 illustrates the STM image of the complex prepared from chloroform. The obtained results confirm the conclusion about the preservation of the compact DNA conformation in chloroform and allow us to propose the toroid-like conformation as the most energetically advantageous one for linear DNA if its charge is neutralized. It should be noted in conclusion that the obtained results testify to the advantages of the use of probe microscopy methods to investigate the complicated complexes of biopolymers and synthetic lipid analogs, in particular the morphology of bioobjects. The abovementioned physical-chemical methods of the investigation of biopolymers in a solution allow us to obtain only an indirect representation of their morphology, whereas STM and AFM reflect the real space structure of the object. The study has been partially supported by Russian Foundation of Basic Researches , projects 96-03-33522a and 97-03-32778a.

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