Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis.

Genome replication induces the generation of large stretches of single-stranded DNA (ssDNA) intermediates that are rapidly protected by single-stranded DNA-binding (SSB) proteins. To date, the mechanism by which tightly bound SSBs are removed from ssDNA by the lagging strand DNA polymerase without compromising the advance of the replication fork remains unresolved. Here, we aimed to address this question by measuring, with optical tweezers, the real-time replication kinetics of the human mitochondrial and bacteriophage T7 DNA polymerases on free-ssDNA, in comparison with ssDNA covered with homologous and non-homologous SSBs under mechanical tension. We find important differences between the force dependencies of the instantaneous replication rates of each polymerase on different substrates. Modeling of the data supports a mechanism in which strong, specific polymerase-SSB interactions, up to ∼12 kBT, are required for the polymerase to dislodge SSB from the template without compromising its instantaneous replication rate, even under stress conditions that may affect SSB-DNA organization and/or polymerase-SSB communication. Upon interaction, the elimination of template secondary structure by SSB binding facilitates the maximum replication rate of the lagging strand polymerase. In contrast, in the absence of polymerase-SSB interactions, SSB poses an effective barrier for the advance of the polymerase, slowing down DNA synthesis.

Dynamics of individual molecular shuttles under mechanical force.

Molecular shuttles are the basis of some of the most advanced synthetic molecular machines. In these devices a macrocycle threaded onto a linear component shuttles between different portions of the thread in response to external stimuli. Here, we use optical tweezers to measure the mechanics and dynamics of individual molecular shuttles in aqueous conditions. Using DNA as a handle and as a single molecule reporter, we measure thousands of individual shuttling events and determine the force-dependent kinetic rates of the macrocycle motion and the main parameters governing the energy landscape of the system. Our findings could open avenues for the real-time characterization of synthetic devices at the single molecule level, and provide crucial information for designing molecular machinery able to operate under physiological conditions.

Single-Stranded Condensation Stochastically Blocks G-Quadruplex Assembly in Human Telomeric RNA.

TERRA is an RNA molecule transcribed from human subtelomeric regions toward chromosome ends potentially involved in regulation of heterochromatin stability, semiconservative replication, and telomerase inhibition, among others. TERRA contains tandem repeats of the sequence GGGUUA, with a strong tendency to fold into a four-stranded arrangement known as a parallel G-quadruplex. Here, we demonstrate by using single-molecule force spectroscopy that this potential is limited by the inherent capacity of RNA to self-associate randomly and further condense into entropically more favorable structures. We stretched RNA constructions with more than four and less than eight hexanucleotide repeats, thus unable to form several G-quadruplexes in tandem, flanked by non-G-rich overhangs of random sequence by optical tweezers on a one by one basis. We found that condensed RNA stochastically blocks G-quadruplex folding pathways with a near 20% probability, a behavior that is not found in DNA analogous molecules.

A Temperature-Jump Optical Trap for Single-Molecule Manipulation.

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability. Our design has features to reduce convection and baseline shifts, which have troubled previous heating-laser instruments. As proof of accuracy, we used the instrument to carry out DNA unzipping experiments in which we derived the average basepair free energy, entropy, and enthalpy of formation of the DNA duplex in a range of temperatures between 5°C and 50°C. We also used the instrument to characterize the temperature-dependent elasticity of single-stranded DNA (ssDNA), where we find a significant condensation plateau at low force and low temperature. Oddly, the persistence length of ssDNA measured at high force seems to increase with temperature, contrary to simple entropic models.

Bond elasticity controls molecular recognition specificity in antibody-antigen binding.

Force-spectroscopy experiments make it possible to characterize single ligand-receptor pairs. Here we measure the spectrum of bond strengths and flexibilities in antibody-antigen interactions using optical tweezers. We characterize the mechanical evolution of polyclonal antibodies generated under infection and the ability of a monoclonal antibody to cross-react against different antigens. Our results suggest that bond flexibility plays a major role in remodeling antibody-antigen bonds in order to improve recognition during the maturation of the humoral immune system.

Electrostatic binding and hydrophobic collapse of peptide-nucleic acid aggregates quantified using force spectroscopy.

Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.

Measurement of work in single-molecule pulling experiments.

A main goal of single-molecule experiments is to evaluate equilibrium free energy differences by applying fluctuation relations to repeated work measurements along irreversible processes. We quantify the error that is made in a free energy estimate by means of the Jarzynski equality when the accumulated work expended on the whole system (including the instrument) is erroneously replaced by the work transferred to the subsystem consisting of the sole molecular construct. We find that the error may be as large as 100%, depending on the number of experiments and on the bandwidth of the data acquisition apparatus. Our theoretical estimate is validated by numerical simulations and pulling experiments on DNA hairpins using optical tweezers.