Mechanical Stability of Single DNA MoleculesHauke Clausen-Schaumann,* Matthias Rief,†Carolin Tolksdorf,* and Hermann E. Gaub**LMV–Mu¨ nchen, Lehrstuhl fu¨ r Angewandte Physik and Center for Nanoscience, 80799 Munich, Germany, and†Department ofBiochemistry B400, Stanford University School of Medicine, Stanford, California 94305-5307 USAABSTRACT Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached toan AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In␭-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition,during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits apronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformationaltransition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionicstrength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, theDNA double helix is considerably destabilized under low salt buffer conditions (ⱕ10 mM NaCl), while high ionic strengthbuffers (1 M NaCl) stabilize the double-helical conformation. The mechanical energy that can be deposited in the DNA doublehelix before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with thebase-pairing free enthalpy ⌬Gbp(T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences againrevealed a close correlation between the mechanical energies at which these sequences melt with base pairing free enthalpies⌬Gbp(sequence): while the melting transition occurs between 65 and 200 pN in␭-phage DNA, depending on the loading rate,the melting transition is shifted to ⬃300 pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA melts at a force of 35 pN.INTRODUCTIONWith the development of new experimental tools allowingpiconewton force resolution and Ångstrøm precision posi-tioning of force sensors, mechanical experiments with sin-gle molecules have become possible (Binnig et al., 1986;Florin et al., 1994; Kasas et al., 1997; Lee et al., 1994;Merkel et al., 1999; Moy et al., 1994; Radmacher et al.,1994; Rief et al., 1997a; Smith et al., 1992). Such experi-ments have not only given new insights into intra- andintermolecular forces; they have also shown variations inphysical parameters of individual molecules with respect tothe mean values derived from ensemble measurements (Per-kins et al., 1997; Smith et al., 1992). Furthermore, byinvestigating single polymers far from their maximum-en-tropy conformation, these experiments have inspired newconcepts in polymer physics, which go far beyond theclassical models of this field and incorporate enthalpicdeformation and conformational transitions of polymers(Ahsan et al., 1998; Heymann and Grubmu¨ller, 1999;Marko, 1997, 1998; Marszalek et al., 1998; Rief et al.,1997b, 1998).Early on, the mechanical properties of DNA attracted theinterest of both physicists and biologists, because of theirimportance to numerous biological processes, such as DNAtranscription, gene expression and regulation, and DNAreplication. Early stretching experiments with single DNAmolecules investigated the influence of electrostatic screen-ing on the persistence length (Smith et al., 1992) as well asthe hydrodynamic coupling between DNA and the sur-rounding fluid (Perkins et al., 1995, 1997). As higher forcescould be applied to the molecule, a highly cooperativeconformational transition was discovered, where the naturalB-DNA is converted into a new overstretched conformationcalled S-DNA (Bensimon et al., 1995; Cluzel et al., 1996;Smith et al., 1996). New theoretical models, as well asmolecular dynamics simulations, have shed light on themolecular details of this overstretching transition (Ahsan etal., 1998; Konrad and Bolonick, 1996; Kosikov et al., 1999;Lebrun and Lavery, 1996; MacKerell and Lee, 1999;Marko, 1997, 1998), and the role of twist stored within thedouble helix has been investigated both experimentally andtheoretically (Allemand et al., 1998; Marko, 1997, 1998;Strick et al., 1996, 1998). More recently, unzipping exper-iments with individual lambda DNA molecules have dem-onstrated a correlation between the unzipping forces and theaverage GC and AT content of the unzipped segment of themolecule (Bockelmann et al., 1997; Essevaz-Roulet et al.,1997), and the unzipping of pure CG and AT sequencesdirectly revealed the sequence-specific base pairing forcesin DNA (Rief et al., 1999). Finally, DNA transcription byRNA polymerase (Wang et al., 1998; Yin et al., 1995) andbinding of RecA to DNA (Hegner et al., 1999; Le´ger et al.,1998) could be directly investigated under native conditionswith single-molecule force experiments.In this study we investigate the mechanical stability ofsingle DNA molecules as a function of stretching velocity,buffer composition, temperature, and DNA sequence, usingAFM-based single molecule force spectroscopy. Further-more, the kinetics of the force-induced melting of the dou-Received for publication 7 September 1999 and in final form 18 November1999.Address reprint requests to Dr. Hermann E. Gaub, Lehrstuhl fu¨r Ange-wandte Physik, Amalienstrasse 54, 80799 Mu¨nchen, Germany. Tel.: ⫹49-89-2180-3172; Fax: ⫹49-89-2180-2050; E-mail: [email protected].© 2000 by the Biophysical Society0006-3495/00/04/1997/11 $2.001997Biophysical Journal Volume 78 April 2000 1997–2007ble helix, as well as its reannealing kinetics, are directlyinvestigated.MATERIALS AND METHODS␭-BstE II digest DNA (length distribution 117-8454 bp) was purchasedfrom Sigma (Deisenhofen, Germany). Duplex poly(dG-dC) (averagelength 1257 bp) and poly(dA-dT) (average length 5090 bp) were purchasedfrom Pharmacia (Freiburg, Germany). For the preparation of dsDNAsamples, the DNA was used as received and diluted with Tris (Sigma)buffer containing 150 mM NaCl, 10 mM Tris (pH 8), 1 mM EDTA(Sigma) to a final concentration of 100␮g/ml. The DNA was allowed toadsorb to a freshly evaporated gold surface from a 100-␮l drop of the 100␮g/ml DNA solution (24-h incubation, ambient temperature), or alterna-tively, a 100-␮l drop of a 100␮g/ml DNA solution was allowed to dry onthe gold surface. Prior to the