A novel procedure for the application of atom probe tomography (APT) to the structural analysis of biological systems, has been recently proposed, whereby the specimen is embedded by a silica matrix and ablated by a pulsed laser source. Such a technique, requires that the silica primer be properly inert and bio-compatible, keeping the native structural features of the system at hand, while condensing into an amorphous, glass-like coating. In this work, we propose a molecular dynamics protocol, aimed at depicting and characterizing the earliest stages of the embedding process of small biomolecules in a solution of water and orthosilicic acid, here, taken as a precursor of the silica matrix. Overall, we observe a negligible influence of orthosilicic acid on the behavior of stable folded systems (such as ubiquitin). Conversely, intrinsically disordered and unstable peptides are affected by the coating, the latter seemingly inhibiting the fluctuations of flexible moieties. While further scrutiny is in order, our assessment offers a first mechanistic insight of the effects of orthosilicic acid, thereby validating its use in the proposed innovative application of APT to the structural resolution of protein molecules.

The cowpea chlorotic mottle virus (CCMV) has emerged as a model system to assess the balance between electrostatic and topological features of single‐stranded RNA viruses, specifically in the context of the viral self‐assembly. Yet, despite its biophysical significance, little structural data on the RNA content of the CCMV virion is available. Here, the conformational dynamics of the RNA2 fragment of CCMV was assessed via coarse‐grained molecular dynamics simulations, employing the oxRNA2 force field. The behavior of RNA2 was characterized both as a freely‐folding molecule and within a mean‐field depiction of the capsid. For the former, the role of the salt concentration, the temperature and of ad hoc constraints on the RNA termini was verified on the equilibrium properties of RNA2. For the latter, a multi‐scale approach was employed to derive a potential profile of the viral cavity from atomistic structures of the CCMV capsid in solution. The conformational ensembles of the encapsidated RNA2 were significantly altered with respect to the freely‐folding counterparts, as shown by the emergence of long‐range motifs and pseudoknots. Finally, the role of the N‐terminal tails of the CCMV subunits is highlighted as a critical feature in the construction of a proper electrostatic model of the CCMV capsid.

The degree of over-/under-winding of the DNA double helix quantified by the superhelical density, is a key feature modulating critical biological processes such as gene expression and regulation: In fact, DNA molecules are able to channel the excess levels of mechanical stress into local defective and denatured states that are promptly detected by, e.g., transcription factors and nuclease enzymes. The occurrence and stability of these motifs is dictated by a complex interplay between topological and sequence-dependent effects, ultimately affecting the global conformational dynamics of the DNA molecule itself. Here, we characterize the impact of the sequence and of the super-helical density on the structural evolution of a 672-bp DNA minicircle via classical molecular dynamics simulations employing the coarse-grained oxDNA force field. We observe that moderately-to-highly undercoiled regimes are associated with the occurrence of stable, some- what broad denaturation bubbles, typically co-localizing with flexible nucleotide sequences on the DNA minicircle: These defects are hardly re-adsorbed by the system, thereby pinning the subsequent dynamics of the molecule. In fact, a similar behavior was recapitulated by enforcing “synthetic”, adjoining DNA mismatches, regardless of the underlying nucleotide sequence, suggesting an effective manner of DNA manipulation.

Ionizing radiation (IR) is a major source of biological hazard, associated with a broad range of detrimental lesions of the structural and molecular integrity of the DNA molecule—often leading to genomic instabilities and severe cellular outcomes. The radiosensitivity of DNA is deeply affected by a variety of chemical and biophysical factors, which control the dynamical behavior of the molecule as well as diverse cellular processes and response pathways. Among these factors, the role of supercoiling in modulating DNA radiosensitivity remains controversial, with the existing literature being inconclusive on its effective contribution. Here, we characterize the linearization of a supercoiled DNA minicircle by double-strand breaks (i.e., the rupture of the covalent DNA backbone on both complementary strands of the double helix) via classical coarse-grained molecular dynamics simulations, and verify how the initial supercoiling regime of the molecule influences the kinetics of the rupturing process. We observe that the excess torsional stress overall enhances the rupturing likelihood but in one specific scenario—associated with a biologically-significant level of (negative) superhelical density: This effect highlights a strong asymmetry between positive and negative supercoiling regimes and provides critical insights on the role of topology on the radiosensitivity of DNA molecules.

Cells and tissue integrity are constantly challenged by the necessity to adapt and respond to mechanical loads. Among cellular components, the nucleus possesses mechano-sensing and mechanotransduction capabilities, yet the molecular mechanisms involved remain poorly defined. Here we investigate whether the mechanical properties of chromatin and its organization into condensates contribute to nuclear adaptation to external forces, while preserving its integrity. By interrogating the effects of MLL4 loss-of-function in Kabuki Syndrome, we find that the balancing of transcriptional and Polycomb condensates tunes nuclear responsiveness to external mechanical forces. MLL4 assembles into mechanosensitive condensates through its prion-like domain, and this response is regulated by the chromatin context. Furthermore, the mechano-sensing activity of MLL4 condensates is instrumental to withstand the physical challenges nuclei experience during cell confinement and migration by preserving their integrity. In Kabuki Syndrome, persistent nuclear envelope rupture triggers cGAS-STING activation, leading to programmed cell death. Together, these findings identify chromatin condensates as active regulators of nuclear mechanosensing and establish a mechanistic link between defective chromatin organization and cGAS–STING activation in Kabuki syndrome.

Objective: Ionizing radiation is a major source of biological hazard, bearing a broad range of detrimental lesions of the DNA structural and molecular integrity - often leading to genomic instabilities and severe cellular outcomes. The sensitivity of the DNA molecule to ionizing radiation is deeply affected by a variety of chemical and biophysical factors controlling its dynamical behavior, as well as by diverse cellular processes and response pathways. With the available literature offering no clear-cut, conclusive perspective, this work aims at characterizing the role of supercoiling in modulating the mechanical response of DNA to double strand breaks, and discuss the outcome within the broader framework of DNA radiosensitivity. Approach: We assess the linearization of a supercoiled 672-bp DNA minicircle by double strand breaks, i.e., the disruption of the covalent DNA backbone on both complementary strands of the double helix, and a fingerprint lesion of ionizing radiation. To this effect, we employ classical coarse-grained molecular dynamics simulations, and verify how the sequence and supercoiling regime of the minicircle affect the kinetics of the rupturing process. Main results: We observe that the excess torsional stress overall enhances the likelihood of the DNA rupturing but in one specific scenario - associated with a biologically-significant level of negative superhelical density - thereby highlighting a strongly non-symmetric behavior between positive and negative supercoiling regimes. Significance: This work deals critical dynamical insights on the role of topology in the mechanical response of DNA to double strand breaks: Together with the decrease of the effective volume of a DNA target enforced by an excess/defect of superhelical density, we infer that a degree of supercoiling belonging to an average biological scenario might factor in the earliest radiobiological response of (naked) DNA.