Research lines

 

The research group led by Dr Felix Ritort investigates the energy of biological processes on the molecular level in the broadest possible sense, from the fundamental understanding of irreversible processes on a microscopic scale through to more advanced applications that allow them to characterise molecular interactions in great detail, measuring energy with a resolution in the tenths of kilocalories per mole (kcal/mol) and statistical information processes down to the tenth of a bit.

 

All this is done with a multidisciplinary approach, combining single-molecule experiments and phenomenological theories in biophysics and fundamental principles of statistical physics applied to non-equilibrium physicochemical systems. The overarching perspective of our research has many applications beyond biophysics, ranging from the thermodynamics and kinetics of small nucleic-acid binding drugs to the aggregation process in macromolecular complexes or the study of the antigen-antibody interactions in the humoral system.

 

Our group is world renowned for having made fundamental advances in the understanding and characterisation of energy folding and assembly of nucleic acids, the study of the kinetics of binding of small ligands to nucleic acids, and the development of fundamental theories that describe the behaviour of small non-equilibrium systems and disordered and glassy systems. Over the coming years, we will continue working on these lines of research that have proven so exciting. Currently we are taking our first steps toward unveiling the fundamental physical principles behind molecular evolution using the single-molecule approach to quantify the energy-information relationship so characteristic of the growing complexity and diversity of populations of molecular mutants. Following is a brief description of each area.

Our group is working on DNA biophysics, molecular folding and energy and information in biophysics

 

DNA biophysics

High-accuracy characterisation of nucleic acid thermodynamics and binding of proteins and peptides to nucleic acids. My lab has shown, for the first time ever, how to combine single DNA unzipping experiments and the finest statistical physics tools to extract the free energy of hybridisation of nucleic acid bases with accuracy on the order of 0.1kcal/mol (or 0.01kT at 298K) [Hug10].

 

Currently we are extending this technique to extract the stacking free energy of different base pairs for DNA under varied conditions of salt and temperature, and for RNA as well. The latter is essential to understanding many regulatory processes inside the cell. With regard to proteins and peptides binding to nucleic acids (or proteins), we will delve deeper into force spectroscopy methods, recently developed in my lab, that are capable of differentiating between non-specific and specific binding (e.g. peptides binding to specific DNA sequences) to extract the affinity and position of binding with high accuracy (tenths of kcal/mol and one base pair resolution) providing a novel, efficient method of molecular footprinting [Cam15].

 

Finally, our recently developed temperature-controlled optical tweezers will allow us to extract the much less-known enthalpies and entropies of hybridisation and binding that are crucial to inferring optimal processing conditions for molecular products (e.g. melting temperatures or denaturant concentrations) [Lor15]. Finally, this technique can be used to study ion competition (e.g. between monovalent and divalent) and the effects of macromolecular crowding or methylation (essential in regulating the genome) on DNA hybridisation.

 

[Hug10] J. M. Huguet, C. V. Bizarro, N. Forns, S. B. Smith, C. Bustamante and F. Ritort, “Single-molecule derivation of salt dependent base-pair free energies in DNA”, Proceedings of the National Academy of Sciences, 107 (2010) 15431-15436

 

[Cam15] J. Camunas-Soler, M. Manosas, S. Frutos, J. Tulla-Puche, F. Albericio and F. Ritort, “Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline”, Nucleic Acids Research, 43 (2015) 2767-2779

 

[Lor15] S. de Lorenzo, M. Ribezzi-Crivellari, R. Arias-Gonzalez, S.B. Smith and F. Ritort, “A Temperature-Jump Optical Trap for Single-Molecule Manipulation”, Biophysical Journal, 108 (2015) 2854-2864 2)


fig1PUBS

Figure 1: Repeated unzipping curves of a 480bp DNA hairpin in the presence of the bis-intercalating peptide Thiocoraline (shown as blue staples in the illustration at the left). The large force peaks along the force-distance curve are indicative of peptide-DNA binding events.

 

Molecular folding

One of the most fascinating non-equilibrium processes in nature is molecular folding or how molecules fold into their natural shape, avoiding the many kinetic traps encountered along the folding pathway and circumventing the so-called Levinthal paradox. With force techniques it is possible to manipulate single DNA, RNA hairpins and proteins by attaching their ends to molecular handles (such as DNA or DNA-RNA hybrid molecules) that are linked to micron-sized beads to control the molecular extension and force in magnetic or optical tweezers.

 

This allows us to monitor in real time how the molecule folds and unfolds by repeatedly moving the trap back and forth. Mechanical force is generally used to induce conformational molecular changes and thus extract thermodynamic binding affinities and kinetic constants for intramolecular and intermolecular reactions. Thus, combining these measurements with theories of statistical physics it is possible to characterise the folding pathway and the molecular free energy landscape of DNA [Ale12], RNAs [Biz12] and proteins [Ale16]. These techniques can be applied to research into other intermolecular interactions, such as antigen-antibody recognition in the humoral system [Ale13]. We are currently extending these studies to design a synthetic funnel for DNA folding.

 

[Ale12] A. Alemany, A. Mossa, I. Junier and F. Ritort, “Experimental free-energy measurements of kinetic molecular states using fluctuation relations”, Nature Physics, 8 (2012) 688-694

 

[Biz12] C. V. Bizarro, A. Alemany and F. Ritort, “Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods”, Nucleic Acids Research, 40 (2012) 6922-6935

 

[Ale16] A. Alemany, B. Rey-Serra, S. Frutos, C. Cecconi and F. Ritort, “Mechanical Folding and Unfolding of Protein Barnase at the Single- Molecule Level”, Biophysical Journal, 110 (2016) 63-74

 

[Ale13] A. Alemany, N. Sanvicens, S. De Lorenzo, P. Marco and F. Ritort, “Bond Elasticity Controls Molecular Recognition Specificity in Antibody-Antigen Binding”, Nano Letters 13 (2013) 5197-5202


fig2PUBS

Figure 2: Folding curves for barnase protein meeasured in passive clamp experiments. Barnase structure in the folded state. The hydrophobic core is shown in blue. From [Ale16]

 

Energy and information in biophysics

Fluctuation theorems (FTs) are fundamental relationships in statistical physics that have provided new methods of extracting free energy differences from non-equilibrium work measurements [Rit08]. Our group has acquired great knowledge applying these methods to the field of molecular biophysics, where knowledge of the free energy of nucleic acid and protein structures is key to understanding their biological function. Originally devised to characterise the free energy of pure equilibrium states, FTs have been extended to accommodate more general situations such akinetic states, systems evolving under feedback control or even glassy systems [Ale15].

 

One of the most exciting outcomes of recent research in this field is the possibility of measuring energy and information at the single-molecule level. The fact that concepts of energy and information are strongly linked in biology is not a surprise to anyone. What is much less evident is how to quantify information, a concept related to statistical entropy in equilibrium thermodynamics that becomes fuzzy in non-equilibrium systems, among which living systems are the most prominent example. Single-molecule experiments provide not only invaluable tools to measure work and energies, but also a suitable playground to better understand the concept and measurement of information.

 

The Maxwell demon, a thought experiment devised by J. C. Maxwell at the end of the 19th century that uses information to violate the second law of thermodynamics, has recently been implemented in the lab using single molecules. Along this line of research, we plan to measure entropy production fluctuations for molecular machines. This is an exciting goal for our lab because the total entropy production in that case is not measurable but can only be inferred under the framework of thermodynamic inference we have recently developed. This is a new field of application for fluctuation theorems where only part of the total entropy production is accessible and the missing part can be inferred by imposing the validity of the fluctuation theorem for the total entropy [Rib14]. One of the most exciting aspects of this topic is the natural emergence of the concept of effective temperature, originally introduced in the context of glassy systems and now directly measurable in single-molecule assays [Die15].

 

Finally, evolutionary ensembles are the most remarkable category of non-equilibrium systems relevant for life. They are characterised by the transduction of available free energy from environmental sources into diversification (information production). Molecular populations of mutants evolving under Darwinian selection produce an evolutionary ensemble characterised by the selective amplification of the fittest species. We have started experiments to quantify the information-content distribution of a mutational ensemble of DNA hairpins under directed molecular evolution and we plan to extend our research in the future to RNA thermosensors and a pleiotropic population of proteins selected by binding either its mRNA or rRNA partners. In this field the right combination of theory and experiments will be essential to uncovering physical principles of evolution based on energy and information measurements.

 

[Rit08] F. Ritort, “Nonequilibrium fluctuations in small systems: from physics to biology”, Advances in Chemical Physics, 137, 31-123 (2008)

 

[Ale15] A. Alemany, M. Ribezzi-Crivellari and F. Ritort, “From free energy measurements to thermodynamic inference in nonequilibrium small systems”, New Journal of Physics, 17 (2015) 075009

 

[Rib14] M. Ribezzi-Crivellari and F. Ritort, “Free-energy inference from partial work measurements in small systems”, Proceedings of the National Academy of Sciences, 111 (2014) E3386-E3394

 

[Die15] E. Dieterich, J. Camunas-Soler, M. Ribezzi-Crivellari, U. Seifert and F. Ritort, “Single-molecule measurement of the effective temperature in non-equilibrium steady states”, Nature Physics, 11 (2015) 971–977


fig3PUBS

Figure 3: Experimental realization of the Maxwell demon in a single DNA hairpin of 20 bp that hops between the folded and unfolded states. At a given time, a measurement is made and, depending on the molecular state, the force is increased or decreased, according to a predetermined protocol (M. Ribezzi-Crivellari and F. Ritort, unpublished)