Resumen de Combining molecular dynamics simulations and atomic force microscopy experiments to rationalize the mechanical properties of double-stranded DNA and RNA
Many biological processes interrogate the mechanical properties of double-stranded DNA (dsDNA) and RNA (dsRNA). Such processes rely on the ability for dsDNA and dsRNA to deform when subjected to mechanical stress or upon interaction with other molecules. From the local motion of individual base pairs upon protein binding, to the global folding of genome-long polymers, these distortions span a wide range of length scales. While immense efforts have been devoted to unveil the mechanical properties of these duplexes, connecting the dynamics at such multiple length scales still remains a challenging problem.
In this PhD dissertation, I present a holistic approach to this problem. I adopt a multiscale perspective, where the level of spatial resolution is thoroughly varied according to the specific question at hand. To that end, I combine atomistic molecular dynamics (MD) simulations, single-molecule experiments, and theoretical models. MD simulations enable exploring the dynamics of the duplexes at the atomic scale and make predictions on how these motions are translated into global, polymer-like mechanical properties. By means of single-molecule techniques I experimentally test some of the computational predictions and measure novel effects that might motivate future theoretical developments. Lastly, simple theoretical models are built to rationalize and bring together the simulation results and the experimental observations.
According to the strategy employed, this Thesis is divided in two parts. In the first part (Chapter 2) I follow a top-down approach. Previous single-molecule measurements performed on long DNA and RNA duplexes motivated the scrutiny of atomistic mechanisms by means of MD simulations. Of particular importance here is the opposite torsional response reported for dsDNA and dsRNA under an external force: dsDNA overwinds when stretched whereas dsRNA unwinds. Guided by this experimental observation, MD simulations reveal an opposite change of the dsDNA and dsRNA interstrand distance upon elongation.
In the second part (Chapters 3 to 6), the strategy followed is predominantly bottom-up: molecular features of particular dsDNA and dsRNA sequences are theoretically predicted (Chapters 3 and 4) or experimentally measured (Chapters 5 and 6) to give rise to certain macroscopic phenomena. In Chapters 3 and 4, I use MD simulations to study the mechanical response of different dsDNA and dsRNA sequences to an external force. In Chapter 3, I unveil that the sequence-dependent dsDNA stretching flexibility is encoded in the shape of the molecule via a structural feature that is named crookedness. In Chapter 4, I report that the nucleotide sequence affects in a strikingly divergent manner the mechanical response of dsRNA and dsDNA. This effect is a consequence of the large flexibility of dsRNA pyrimidine-purine steps. In Chapters 5 and 6 I dwell on two peculiar kinds of sequences: dsDNA A-tracts and dsRNA AU-tracts. The first kind is known to introduce a bend in the DNA and has been well characterized at the local level, while its global mechanical properties have remained controversial. In Chapter 5, I build upon the well-known local features of A-tracts to provide a comprehensive description of their global flexibility, disentangling the different mechanical properties of these sequences by means of a combination of single-molecule techniques. Motivated by the dsDNA A-tracts, I report in Chapter 6 a systematic study of sequence-induced bending in dsRNA. Firstly, MD simulations are used to identify a sequence motif, which are named AU-tract, which stabilizes bent conformations in dsRNA. This computational prediction is then experimentally demonstrated by atomic force microscopy (AFM) imaging, which reveals that dsRNA molecules rich in AU-tracts are more prone to adopt bent conformations than control dsRNA molecules of arbitrary sequence.
Through the examples presented, this Thesis highlights how the combination of MD simulations and single-molecule techniques can contribute towards bridging the gap between the different length scales involved in the mechanical properties of dsDNA and dsRNA.
