3.2 Mechanical unzipping of DNA

In a very simplified view of life, all cells react to external impulses (food, heat) by performing some specific tasks (digest, crawl). All these specific tasks are carried out by proteins (enzymes, molecular motors). So cells need to continuously access to the genetic information stored in the sequence of DNA to synthesize proteins. Cell division is another example of a process that requires to access to the genetic information. In order to read the DNA sequence, the two strands of DNA must be split apart so that the cellular machinery can interact with the bases that code the instructions. For instance during replication, helicases are the proteins responsible of separating the two strands of DNA. The process of strand separation carried out by proteins was reproduced in vitro in the last decades of the 20th century [100]. Recently it has been studied at the single-molecule level [101,102,103].

The strand separation of DNA can also be produced in vitro without the proteins of the cell. This can be achieved by using an external agent such as temperature, chemical agents or force. Mechanical melting is a process that consists in pulling apart (i.e., unzipping) the two strands of a double-stranded DNA (dsDNA) molecule until the base pairs that hold the DNA duplex together are disrupted and two single-stranded DNA (ssDNA) molecules are obtained. The term unzipping presumes that the DNA molecule is pulled from the $ 3'$ and the $ 5'$ extremities of one end of the molecule. The process of unzipping has its opposite: the rezipping. Indeed when the force is released, the DNA molecule tends to return to its native state, i.e., completely folded.

The mechanical separation of DNA was initially explored by pulling the DNA from its $ 5'$ extremities using an Atomic Force Microscopy (AFM) [104,105]. The shear stress induced on the strands produced sudden disruptions of the base-pairs. The first unzipping of single molecules of DNA was carried out by Bockelmann and coworkers in 1997 using microneedles [106,21,107]. Inspired by a previous experiment [108], they tethered the DNA molecule between a coverslip and a movable glass microneedle, which allowed to measure the force by calibrating the deformation of the microneedle (see Fig. 3.6a). Mechanical unzipping was monitored by simultaneously measuring the displacement of the glass microneedle and the force applied on the molecule. The resulting force vs. extension curve (FEC) revealed a reproducible pattern which was correlated with the local content of Guanine and Cytosine (GC) along the DNA sequence. In particular, more force was required to unzip regions with high GC content as compared to regions with high AT content. Besides, the energies measured were compatible with the melting experiments performed in bulk [109]. Latter, Rief et al. were able to produce the overstretching and the unzipping of DNA in the same experiment using AFM (see Fig. 3.6b) [22,110]. These experiments were a direct proof that the base-pairing forces in DNA were sequence specific. In 2001, Liphardt et al. [49] unzipped short RNA hairpins and observed coexistence and hopping between folded and unfolded states using optical tweezers. In a new improved experiment (see Fig. 3.6c), Bockelmann et al. repeated the DNA unzipping with optical tweezers [24]. Optical tweezers provided much more spatial and temporal resolution as well as a higher trap stiffness compared to glass microneedles. The unzipping experiments showed hopping between intermediate states and the experimental FEC was qualitatively reproduced by a mesoscopic model based on the Nearest-Neighbor model for nucleic acids [27,26]. Still, the unzipping/rezipping curves showed some irreproducibility due to experimental drift, non-equilibrium effects (hysteresis) and statistical variation. A similar work done by the same group [111] showed that the unzipping experiments performed at high pulling speed deviated significantly from the quasistatic ones. They attributed such deviation to rotational drag effects. In 2003, Danilowicz et al. [23] unzipped DNA at constant force using magnetic tweezers (see Fig. 3.6d). The process showed a succession of breakages of base-pairs, typical of first order phase transitions. Since then, unzipping experiments have been the focus of attention of some studies [112,113]. Unzipping has also been used as a tool to explore other processes of the cell such as the protein-DNA interaction [114] or the translation of proteins observed in single ribosomes [115]. Besides, the theoretical studies have also focused their attention on different particular aspects of the unzipping experiments such as the scaling properties [29], the kinetics [116] or the problem of sequencing DNA by force [117].

Figure 3.6: Unzipping of DNA with different techniques. (a) Microneedles [106]. (b) AFM [22]. (c) Optical tweezers [24]. (d) Magnetic tweezers [23].
\includegraphics[width=\textwidth]{figs/chapter3/unzippings.eps}



Subsections
JM Huguet 2014-02-12