We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage DNA. The 3 and 5 extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected. Mechanical force at the molecular level is involved in the action of many enzymes. This is the case for the processes of replication or transcription in which enzymes translocate processively with respect to DNA. Such translocation occurs unidirectionally over long segments of DNA, and the enzymatic machinery has to develop a force against a number of impediments: the disruption of complementary base pairs, the possible attachments of the DNA or the enzymes to cellular components, structural proteins that coat DNA and have to be displaced, topological constraint, and viscous friction. The force necessary to stop a transcribing Escherichia coli polymerase recently has been measured (1). In the case of replication (2), the DNA double helix is opened, and two daughter strands are formed. The opening may be associated to the translocation mechanism of the polymerase itself or may be assisted by accessory proteins like helicases or single-strand binding proteins. Moreover, because the strands are intertwined, strand separation is coupled to a local rotation. Topological constraints are resolved by topoisomerases (3, 4).Long before the enzymes associated to replication were known, a simple model had been considered (5) in which the mechanism of unwinding the strands during replication is coupled to rotation of the whole molecule. A molecular configuration with a Y shape had been proposed in which the vertical part is the parent helix, and the two arms are the separated strands that get replicated. As replication proceeds, a ''speedometer cable'' rotation motion was proposed for all three branches of the Y.We have set up an experiment to measure directly the forces involved in the elementary process of mechanical strand separation, with no enzyme present. The experiment presented here is approaching the Levinthal and Crane (5) configuration.Force measurement on single molecules of DNA is an emerging field (6-13). For typical molecular interactions involving biomolecules, the forces involved are small (sub-picoNewton to 10s of picoNewtons [pN ϭ 10 Ϫ12 N]). For this reason, sensitive measuring devices (14-17) such as optical tweezers, soft microneedles, or levers of atomic force microscopes have been used.

Force measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system.

We have pulled apart the two strands of a DNA double helix. The forces measured during this process show a sequence specific variation on the piconewton scale. Opening two helical molecules with the same sequence from opposite sides gives two signatures which are not simply related by symmetry. In a theoretical model, this is explained as a molecular stick-slip motion which does not involve instabilities and is determined by the sequence. [S0031-9007(97)04560-2]

We have separately attached the two complementary strands of one end of a DNA double helix to a glass slide and a glass microneedle. Displacing the slide away from the needle, the molecule is progressively pulled open and the changing deflection of the needle gives the corresponding variation in the opening force. Force signals which are very rich in detail are reproducibly obtained. The average level and amplitude of the force signal is almost independent of the opening velocity in the interval 20 nm/s to 800 nm/s. A theoretical description based on the assumption of thermal equilibrium allows us to link the measured force curves to the genomic sequence of the DNA. A molecular stick-slip motion is revealed, which in contrast to the dynamics of macroscopic solid friction is a deterministic and reproducible process. This process is considered experimentally and theoretically.

In this paper we summarize part of our work on the mechanical unzipping of DNA. We have prepared molecular constructions which allow us to attach the two complementary strands of one end of a single DNA molecule of the bacteriophage λ separately to a glass microscope slide and a microscopic bead. In a first series of experiments, a soft microneedle acting as a force sensor is attached to the bead and its deflection is measured with an optical microscope. In a second series, we use an optical trapping interferometer to capture the bead and to measure its displacement to nm resolution. The sample is slowly displaced with respect to the force measurement device, leading to a progressive opening of the double helix. The force measured during this mechanical opening shows a characteristic variation which is related to the base pair sequence of the DNA molecule. To cite this article: U. Bockelmann et al., C. R. Physique 3 (2002) 585-594.  2002 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS micromanipulation / optical tweezers / DNA unzipping Ouverture mécanique de la molécule d'ADN par micro-manipulation et mesure de force Résumé Dans cet article nous résumons une partie de notre travail sur l'ouverture mécanique de l'ADN. Nous avons préparé des constructions moléculaires qui nous permettent d'attacher les deux brins complémentaires d'une extrémité d'une molécule unique d'ADN du bactériophage λ, séparément à une lamelle de microscope et à une bille microscopique. Dans une première série d'expériences, un levier souple de verre, agissant comme capteur de force, est attaché à la bille et sa deflexion est mesurée avec un microscope optique. Dans une seconde série, nous utilisons un piège optique interférométrique pour capturer la bille et mesurer son déplacement avec une résolution nanométrique. L'échantillon est déplacé lentement par rapport au système de mesure de force, induisant une ouverture progressive de la double hélice. La force, mesurée pendant cette ouverture mécanique, montre une variation caractéristique qui est reliée à la séquence des paires de base de la molécule d'ADN. Pour citer cet article : U. Bockelmann et al., C. R. Physique 3 (2002) 585-594.  2002 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS micromanipulation / pinces optiques / ouverture de l'ADN * Correspondence and reprints.