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Phosphorothioates (PS) have proven their effectiveness in the area of therapeutic oligonucleotides with applications spanning from cancer treatment to neurodegenerative disorders. Initially, PS substitution was introduced for the antisense oligonucleotides (PS ASOs) because it confers an increased nuclease resistance meanwhile ameliorates cellular uptake and in-vivo bioavailability. Thus, PS oligonucleotides have been elevated to a fundamental asset in the realm of gene silencing therapeutic methodologies. But, despite their wide use, little is known on the possibly different structural changes PS-substitutions may provoke in DNA·RNA hybrids. Additionally, scarce information and significant controversy exists on the role of phosphorothioate chirality in modulating PS properties. Here, through comprehensive computational investigations and experimental measurements, we shed light on the impact of PS chirality in DNA-based antisense oligonucleotides; how the different phosphorothioate diastereomers impact DNA topology, stability and flexibility to ultimately disclose pro-Sp S and pro-Rp S roles at the catalytic core of DNA Exonuclease and Human Ribonuclease H; two major obstacles in ASOs-based therapies. Altogether, our results provide full-atom and mechanistic insights on the structural aberrations PS-substitutions provoke and explain the origin of nuclease resistance PS-linkages confer to DNA·RNA hybrids; crucial information to improve current ASOs-based therapies.

Exascale computing has been a dream for ages and is close to becoming a reality that will impact how molecular simulations are being performed, as well as the quantity and quality of the information derived for them. We review how the biomolecular simulations field is anticipating these new architectures, making emphasis on recent work from groups in the BioExcel Center of Excellence for High Performance Computing. We exemplified the power of these simulation strategies with the work done by the HPC simulation community to fight Covid-19 pandemics.

Exascale computing has been a dream for ages and is close to becoming a reality that will impact how molecular simulations are being performed, as well as the quantity and quality of the information derived for them. We review how the biomolecular simulations field is anticipating these new architectures, making emphasis on recent work from groups in the BioExcel Center of Excel-lence for High Performance Computing. We exemplified the power of these simulation strategies with the work done by the HPC simulation community to fight Covid-19 pandemics., European Commission (BioExcel-2project), Grant/Award Number: 823830; Instituto de Salud Carlos III, Grant/AwardNumber: PT17/0009/0007; Ministerio de Ciencia e Innovación, Grant/Award Numbers: PID2020-116620GB-I00, RTI2018-096704-B-100., Peer Reviewed, Postprint (author's final draft)

We analyze the physical origin and the chemical and biological consequences of the asymmetry that occurs in DNA·RNA hybrids when the purine/pyrimidine (Pu/Py) ratio is different in the DNA and RNA strands. When the DNA strand of the hybrid is Py rich, the duplex is much more stable, rigid, and A-like than when the DNA strand is Pu rich. The origins of this dramatic asymmetry are double: first, the apparently innocuous substitution dT → rU produces a significant decrease in stacking, and second, backbone distortions are larger for DNA(Pu)·RNA(Py) hybrids than for the mirror RNA(Pu)·DNA(Py) ones. The functional impact of the structural and dynamic asymmetry in the biological activities of hybrids is dramatic and can be used to improve the efficiency of antisense-type strategies on the basis of the degradation of hybrids by RNase H or gene editing using CRISPR-Cas9 technology. RNA·DNA hybrids are not exotic structures created in the laboratory but rather duplexes showing a wide range of biological functions and exhibiting incredible potential in gene therapies, such as those based on RNAse H antisense and CRISPR-Cas9 gene-editing approaches. These duplexes show an intrinsic asymmetry that can represent a crucial role for biological function and biotechnological applications. By combining a large set of theoretical, chemical, biophysical, bioinformatics, and molecular biology methods, we describe and decipher the origins and biological consequence (in terms of RNAse H and CRISPR-Cas9 susceptibility) of the asymmetry generated in hybrids when the Pur/Pyr ratio is significantly different in DNA and RNA strands. Our work not only unveils the basic physicochemical properties at the basis of RNA·DNA asymmetry but also significantly contributes to the development of more efficient hybrid-based therapies with clear biotechnological and biomedical implications. RNA·DNA hybrids present an intrinsic structural, dynamic, and energetic asymmetry that depends on the Pur or Pyr natur

Nucleic acid polymerization is a key process for genetic inheritance in all living cells. This is performed by a set of DNA/RNA polymerases (Pols) that are effective drug targets in nove therapies. However, DNA damages represent an obstacle for the replication machinery. Here, the role of trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), stand out. Pol-η bypasses ultraviolet-induced basing its function on a highly flexible and conserved R61 and on a transient third ion resolved at the catalytic site in post-reactive state. Nevertheless, how these element assist damaged-DNA replication is still poorly understood. We unravel a highly cooperative mechanism for DNA repair performed by human Pol-η that, via specific R61 conformations, assists the recruitment of the (nucleotide-triphosphate) dNTP in pre-reactive state and ehnances pyrophosphate leaving in post-reactive state. Moreover, bioinformatics analysis revealed that the dNTP always forms an intramolecular H-bond upon formation of the Michaelis-Menten complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and nucleic acid translocation. Thus, SAM allows an elegant closed-loop sequence of chemical and physical steps for Pols catalysis. Our proposed mechanism is corroborated via ab initio QM/MM simulations on Pol-η. The structural conservation of DNA/RNA Pols supports the extension of SAM to all Pols. Finally, we identified key amino-acids and cations optimally placed nearby the active site of two-metal-ion enzymes and ribozymes such as group-II intron. Such elements interact with the reactants and orient the substrates into the active site, being therefore indispensable for catalysis. Our analysis suggests an unprecedented extension of the two-metal-ion architecture in DNA and RNA polymerases, nucleases and ribozymes. In spite of different biopolymer scaffolds, si