1.3 Single-molecule techniques

Traditionally, the experiments in chemistry and biology have been performed in bulk. Bulk experiments involve a large amount of substance (moles, grams, milliliters, etc.). Whatever the experiment is (reaction, measurement, detection), it is performed on a large collection of molecules. These experiments show the average behavior of the molecules and they have been used to set the basis of experimental sciences for centuries.

The detection of single particles has been pursuit by scientists since John Dalton proposed the modern atomic-molecular theory of matter in 1808. Physicists have been able to detect photons, electrons and sub-atomic particles using sensitive detectors (photomultipliers, particle accelerators, etc.). However, some of the most interesting phenomena in physics are cooperative and involve lots of particles: phase transitions, Bose-Einstein condensates. So single-molecule techniques might seem that have little to offer when studying elementary particles or molecules.

However, biomolecules have internal structure and they show complex behavior. So the study of biomolecules benefits from single-molecule techniques [42]. The key point here is that matter is viewed as the result of gathering complex individuals. Using single-molecule experiments (SME) the individual molecules can be manipulated an measured one at a time. The advantage is that the information is not averaged. Instead, one can measure deviations from the average bulk behavior and probability distributions. For instance, biophysicists have been able to measure the step size distribution of molecular motors; the energy consumption of enzymes; and the kinetics of biochemical reactions [33]. All these new amount of information complements the traditional bulk assays.

The first SME in biophysics were performed in the early 1990's [43]. Block et al. were able to characterize the movement of kinesin (i.e., a molecular motor in charge of transportation of substances within the cell) at the single molecule level using optical tweezers [44]. Later, Finer et al. described the working of myosin, another molecular motor that drives muscular contraction [45]. The SME of other molecular motors like F$ _1$-ATPase [46] or RNA polymerase [47] were extensively studied. Among other properties of the motors, these works measured the step size, the stall force or the efficiency at different ATP concentrations. All these magnitudes are hardly measured in bulk. Other SME experiments [2] have focused on the elongational [48] and torsional [33] elasticity of single DNA molecules and the folding/unfolding kinetics of RNA [49] and proteins [50].

The development of SME have pushed the instrumentation forward. The technical devices related with positioning (piezoelectric crystals, motor driven stages), detection (position sensitive detectors, high frequency cameras) and transduction (nanometric cantilevers, magnetic microspheres) have greatly expanded in the last years. Biochemistry has also standardized protocols and reactions commonly used in SME (labeling kits, fluorescent tags). The most productive single-molecule techniques are single-molecule fluorescence, Atomic Force Microscopy (AFM), Magnetic Tweezers (MT) and Laser Optical Tweezers (LOT). Figure 1.3 shows a brief description of them.

Figure 1.3: Single-molecule techniques. (a) Fluorescence resonance energy transfer (FRET). FRET is based on the transmission of energy between fluorescent dyes (donor and acceptor). The efficiency of such transmission depends on the distance between the dyes. It can be used to detect the folding/unfolding of a molecule. (b) AFM. AFM produces and measures the forces applied on a molecule (depicted in green). The force applied is directly related to the bending of a solid state cantilever, whose elasticity is known. The bending of the cantilever is measured from the deflection of a laser beam reflected on the cantilever. (c) MT. The molecule is attached between a magnetic bead and the surface of a cover glass. MT apply forces by immersing the bead in a magnetic field gradient. The force is measured by determining the position of the bead. The magnetic field is produced by magnets that can be moved and rotated to exert force and torque on the molecule. (d) LOT. LOT exerts forces on dielectric beads by using the pressure of radiation of a laser beam. A detailed description will be given in chapter 2.

SME are continuously evolving. The original pioneering experiments can be reproduced by most labs and such experiments have become starting points to study more complex systems. The current trend is to improve and combine techniques and perform several single-molecule experiments in parallel. For instance, optical tweezers initially had one trap. Nowadays, most of the new constructed optical tweezers have two or more traps [51].

The research in molecular biophysics is definitely tied to single-molecule techniques.

JM Huguet 2014-02-12