A Review on Small Organic Colorimetric and Fluorescent Hosts for the Detection of Cobalt and Nickel Ion.

In recent years, there has been a notable increase in efforts to advance efficient hosts for detecting cobalt and nickel ions, driven by their extensive industrial applications and environmental significance. This review meticulously examines the progress made in small organic colorimetric and fluorescent hosts tailored specifically for the sensitive and selective detection of cobalt and nickel ions. It delves into a diverse range of molecular architectures, including organic ligands, elucidating their unique attributes such as sensitivity, selectivity, and response time. Moreover, the review precisely explores the underlying principles governing the colorimetric and fluorescent mechanisms employed by these hosts, shedding light on the intricate interactions between the sensing moieties and the target metal ions. Furthermore, it critically evaluates the practical applicability of these hosts, considering crucial factors such as detection limits, recyclability, and compatibility with complex sample matrices. Additionally, exploration extends to potential challenges and prospects in the field, emphasizing the imperative for ongoing innovation to address emerging environmental and analytical demands. Eventually, through this comprehensive examination, the review seeks to contribute to the ongoing endeavor to develop robust and efficient tools for monitoring and detecting cobalt and nickel metal ions in diverse analytical scenarios.

The Folding of Germ Granule mRNAs Controls Intermolecular Base Pairing in Germ Granules and Maintains Normal Fly Development.

Drosophila germ granules enrich mRNAs critical for fly development. Within germ granules, mRNAs form multi-transcript clusters marked by increased mRNA concentration, creating an elevated potential for intermolecular base pairing. However, the type and abundance of intermolecular base pairing in mRNA clusters is poorly characterized. Using single-molecule super-resolution microscopy, chemical probing for base accessibility, phase separation assays, and simulations, we demonstrated that mRNAs remain well-folded upon localization to germ granules. While most base pairing is intramolecular, mRNAs still display the ability for intermolecular base pairing, facilitating clustering without high sequence complementarity or significant melting of secondary structure. This base pairing among mRNAs is driven by scattered and discontinuous stretches of bases appearing on the surface of folded RNAs, providing multivalency to clustering but exhibits low probability for sustained interactions. Notably, engineered germ granule mRNAs with exposed GC-rich complementary sequences (CSs) presented within stable stem loops induce sustained base pairing in vitro and enhanced intermolecular interactions in vivo. However, the presence of these stem loops alone disrupts fly development, and the addition of GC-rich CSs exacerbates this phenotype. Although germ granule mRNAs contain numerous GC-rich CSs capable of stable intermolecular base pairing, they are primarily embedded by RNA folding. This study emphasizes the role of RNA folding in controlling the type and abundance of intermolecular base pairing, thereby preserving the functional integrity of mRNAs within the germ granules.

Salt-Dependent Self-Association of Trinucleotide Repeat RNA Sequences.

Repeat RNA sequences self-associate to form condensates. Simulations of a coarse-grained single-interaction site model for (CAG)n (n = 30 and 31) show that the salt-dependent free energy gap, ΔGS, between the ground (perfect hairpin) and the excited state (slipped hairpin (SH) with one CAG overhang) of the monomer for (n even) is the primary factor that determines the rates and yield of self-assembly. For odd n, the free energy (GS) of the ground state, which is an SH, is used to predict the self-association kinetics. As the monovalent salt concentration, CS, increases, ΔGS and GS increase, which decreases the rates of dimer formation. In contrast, ΔGS for shuffled sequences, with the same length and sequence composition as (CAG)31, is larger, which suppresses their propensities to aggregate. Although demonstrated explicitly for (CAG) polymers, the finding of inverse correlation between the free energy gap and RNA aggregation is general.

Transcription-induced active forces suppress chromatin motion.

The organization of interphase chromosomes in a number of species is starting to emerge thanks to advances in a variety of experimental techniques. However, much less is known about the dynamics, especially in the functional states of chromatin. Some experiments have shown that the motility of individual loci in human interphase chromosome decreases during transcription and increases upon inhibiting transcription. This is a counterintuitive finding because it is thought that the active mechanical force (F) on the order of ten piconewtons, generated by RNA polymerase II (RNAPII) that is presumably transmitted to the gene-rich region of the chromatin, would render it more open, thus enhancing the mobility. We developed a minimal active copolymer model for interphase chromosomes to investigate how F affects the dynamical properties of chromatin. The movements of the loci in the gene-rich region are suppressed in an intermediate range of F and are enhanced at small F values, which has also been observed in experiments. In the intermediate F, the bond length between consecutive loci increases, becoming commensurate with the distance at the minimum of the attractive interaction between nonbonded loci. This results in a transient disorder-to-order transition, leading to a decreased mobility during transcription. Strikingly, the F-dependent change in the locus dynamics preserves the organization of the chromosome at [Formula: see text]. Transient ordering of the loci, which is not found in the polymers with random epigenetic profiles, in the gene-rich region might be a plausible mechanism for nucleating a dynamic network involving transcription factors, RNAPII, and chromatin.

Structural basis for the preservation of a subset of topologically associating domains in interphase chromosomes upon cohesin depletion.

Compartment formation in interphase chromosomes is a result of spatial segregation between euchromatin and heterochromatin on a few megabase pairs (Mbp) scale. On the sub-Mbp scales, topologically associating domains (TADs) appear as interacting domains along the diagonal in the ensemble averaged Hi-C contact map. Hi-C experiments showed that most of the TADs vanish upon deleting cohesin, while the compartment structure is maintained, and perhaps even enhanced. However, closer inspection of the data reveals that a non-negligible fraction of TADs is preserved (P-TADs) after cohesin loss. Imaging experiments show that, at the single-cell level, TAD-like structures are present even without cohesin. To provide a structural basis for these findings, we first used polymer simulations to show that certain TADs with epigenetic switches across their boundaries survive after depletion of loops. More importantly, the three-dimensional structures show that many of the P-TADs have sharp physical boundaries. Informed by the simulations, we analyzed the Hi-C maps (with and without cohesin) in mouse liver and human colorectal carcinoma cell lines, which affirmed that epigenetic switches and physical boundaries (calculated using the predicted 3D structures using the data-driven HIPPS method that uses Hi-C as the input) explain the origin of the P-TADs. Single-cell structures display TAD-like features in the absence of cohesin that are remarkably similar to the findings in imaging experiments. Some P-TADs, with physical boundaries, are relevant to the retention of enhancer-promoter/promoter-promoter interactions. Overall, our study shows that preservation of a subset of TADs upon removing cohesin is a robust phenomenon that is valid across multiple cell lines.

Competition between Stacking and Divalent Cation-Mediated Electrostatic Interactions Determines the Conformations of Short DNA Sequences.

Interplay between divalent cations (Mg2+ and Ca2+) and single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), as well as stacking interactions, is important in nucleosome stability and phase separation in nucleic acids. Quantitative techniques accounting for ion-DNA interactions are needed to obtain insights into these and related problems. Toward this end, we created a sequence-dependent computational TIS-ION model that explicitly accounts for monovalent and divalent ions. Simulations of the rigid 24 base-pair (bp) dsDNA and flexible ssDNA sequences, dT30 and dA30, with varying amounts of the divalent cations show that the calculated excess number of ions around the dsDNA and ssDNA agree quantitatively with ion-counting experiments. Using an ensemble of all-atom structures generated from coarse-grained simulations, we calculated the small-angle X-ray scattering profiles, which are in excellent agreement with experiments. Although ion-counting experiments mask the differences between Mg2+ and Ca2+, we find that Mg2+ binds to the minor grooves and phosphate groups, whereas Ca2+ binds specifically to the minor groove. Both Mg2+ and Ca2+ exhibit a tendency to bind to the minor groove of DNA as opposed to the major groove. The dA30 conformations are dominated by stacking interactions, resulting in structures with considerable helical order. The near cancellation of the favorable stacking and unfavorable electrostatic interactions leads to dT30 populating an ensemble of heterogeneous conformations. The successful applications of the TIS-ION model are poised to confront many problems in DNA biophysics.

Brewing COFFEE: A Sequence-Specific Coarse-Grained Energy Function for Simulations of DNA-Protein Complexes.

DNA-protein interactions are pervasive in a number of biophysical processes ranging from transcription and gene expression to chromosome folding. To describe the structural and dynamic properties underlying these processes accurately, it is important to create transferable computational models. Toward this end, we introduce Coarse-grained Force Field for Energy Estimation, COFFEE, a robust framework for simulating DNA-protein complexes. To brew COFFEE, we integrated the energy function in the self-organized polymer model with side-chains for proteins and the three interaction site model for DNA in a modular fashion, without recalibrating any of the parameters in the original force-fields. A unique feature of COFFEE is that it describes sequence-specific DNA-protein interactions using a statistical potential (SP) derived from a data set of high-resolution crystal structures. The only parameter in COFFEE is the strength (λDNAPRO) of the DNA-protein contact potential. For an optimal choice of λDNAPRO, the crystallographic B-factors for DNA-protein complexes with varying sizes and topologies are quantitatively reproduced. Without any further readjustments to the force-field parameters, COFFEE predicts scattering profiles that are in quantitative agreement with small-angle X-ray scattering experiments, as well as chemical shifts that are consistent with NMR. We also show that COFFEE accurately describes the salt-induced unraveling of nucleosomes. Strikingly, our nucleosome simulations explain the destabilization effect of ARG to LYS mutations, which do not alter the balance of electrostatic interactions but affect chemical interactions in subtle ways. The range of applications attests to the transferability of COFFEE, and we anticipate that it would be a promising framework for simulating DNA-protein complexes at the molecular length-scale.

Free volume theory explains the unusual behavior of viscosity in a non-confluent tissue during morphogenesis.

A recent experiment on zebrafish blastoderm morphogenesis showed that the viscosity (η) of a non-confluent embryonic tissue grows sharply until a critical cell packing fraction (ϕS). The increase in η up to ϕS is similar to the behavior observed in several glass-forming materials, which suggests that the cell dynamics is sluggish or glass-like. Surprisingly, η is a constant above ϕS. To determine the mechanism of this unusual dependence of η on ϕ, we performed extensive simulations using an agent-based model of a dense non-confluent two-dimensional tissue. We show that polydispersity in the cell size, and the propensity of the cells to deform, results in the saturation of the available free area per cell beyond a critical packing fraction. Saturation in the free space not only explains the viscosity plateau above ϕS but also provides a relationship between equilibrium geometrical packing to the dramatic increase in the relaxation dynamics.

Scaling regimes for wormlike chains confined to cylindrical surfaces under tension.

We compute the free energy of confinement [Formula: see text] for a wormlike chain (WLC), with persistence length [Formula: see text], that is confined to the surface of a cylinder of radius R under an external tension f using a mean field variational approach. For long chains, we analytically determine the behavior of the chain in a variety of regimes, which are demarcated by the interplay of [Formula: see text], the Odijk deflection length ([Formula: see text]), and the Pincus length ([Formula: see text], with [Formula: see text] being the thermal energy). The theory accurately reproduces the Odijk scaling for strongly confined chains at [Formula: see text], with [Formula: see text]. For moderate values of f, the Odijk scaling is discernible only when [Formula: see text] for strongly confined chains. Confinement does not significantly alter the scaling of the mean extension for sufficiently high tension. The theory is used to estimate unwrapping forces for DNA from nucleosomes.

Conformational fluctuations and phases in fused in sarcoma (FUS) low-complexity domain.

The well-known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA-binding protein, Fused in Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low-complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid-state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril structure (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C-terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site-specific. Simulations show that phosphorylation of residues within the fibril has a greater destabilization effect than residues that are outside the fibril region, which accords well with experiments. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular explanation.

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

Observe without disturbing: tracer particles sense local stresses in cell collectives without affecting the cancer cell dynamics.

Measurements of local stresses on the cancer cells (CCs), inferred by embedding inert compressible tracer particles (TPs) in a growing multicellular spheroid (MCS), show that pressure decreases monotonically as the distance from the core of the MCS increases. How faithfully do the TPs report the local stresses in the CCs is an important question because pressure buildup in the MCS is dynamically generated due to CC division, which implies that the CC dynamics should be minimally altered by the TPs. Here using theory and simulations, we show that although the TP dynamics is unusual, exhibiting sub-diffusive behavior on times less than the CC division times and hyper-diffusive dynamics in the long-time limit, they do not affect the long-time CC dynamics. The CC pressure profile within the MCS, which decays from a high value at the core to the periphery, is almost identical with and without the TPs. That the TPs have a small effect on the local stresses in the MCS implies that they are reasonale reporters of the CC microenvironment.

Brewing COFFEE: A sequence-specific coarse-grained energy function for simulations of DNA-protein complexes.

DNA-protein interactions are pervasive in a number of biophysical processes ranging from transcription, gene expression, to chromosome folding. To describe the structural and dynamic properties underlying these processes accurately, it is important to create transferable computational models. Toward this end, we introduce Coarse grained force field for energy estimation, COFFEE, a robust framework for simulating DNA-protein complexes. To brew COFFEE, we integrated the energy function in the Self-Organized Polymer model with Side Chains for proteins and the Three Interaction Site model for DNA in a modular fashion, without re-calibrating any of the parameters in the original force-fields. A unique feature of COFFEE is that it describes sequence-specific DNA-protein interactions using a statistical potential (SP) derived from a dataset of high-resolution crystal structures. The only parameter in COFFEE is the strength (λDNAPRO) of the DNA-protein contact potential. For an optimal choice of λDNAPRO, the crystallographic B-factors for DNA-protein complexes, with varying sizes and topologies, are quantitatively reproduced. Without any further readjustments to the force-field parameters, COFFEE predicts the scattering profiles that are in quantitative agreement with SAXS experiments as well as chemical shifts that are consistent with NMR. We also show that COFFEE accurately describes the salt-induced unraveling of nucleosomes. Strikingly, our nucleosome simulations explain the destabilization effect of ARG to LYS mutations, which does not alter the balance of electrostatic interactions, but affects chemical interactions in subtle ways. The range of applications attests to the transferability of COFFEE, and we anticipate that it would be a promising framework for simulating DNA-protein complexes at the molecular length-scale.

Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation.

Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid-liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA-RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum-which depends on the exact sequence, n, and ionic conditions-is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Entropic contribution of ACE2 glycans to RBD binding.

The spike protein of the SARS-CoV-2 virus (the causative agent of COVID-19) recognizes the host cell by binding to the peptidase domain (PD) of the extracellular receptor angiotensin-converting enzyme 2 (ACE2). A variety of carbohydrates could be attached to the six asparagines in the PD, resulting in a heterogeneous population of ACE2 glycoforms. Experiments have shown that the binding affinity of glycosylated and deglycosylated ACE2 to the virus is virtually identical. In most cases, the reduction in glycan size correlates with stronger binding, which suggests that volume exclusion, and hence entropic forces, determine the binding affinity. Here, we quantitatively test the entropy-based hypothesis by developing a lattice model for the complex between ACE2 and the SARS-CoV-2 spike protein receptor-binding domain (RBD). Glycans are treated as branched polymers with only volume exclusion, which we justify using all-atom molecular dynamics simulations in explicit water. We show that the experimentally measured changes in the ACE2-RBD dissociation constants for a variety of engineered ACE2 glycoforms are in reasonable agreement with our theory, thus supporting our hypothesis. However, a quantitative recovery of all the experimental data could require weak attractive interactions.

Structural changes in chromosomes driven by multiple condensin motors during mitosis.

We create a computational framework that utilizes loop extrusion (LE) by multiple condensin I/II motors to predict changes in chromosome organization during mitosis. The theory accurately reproduces the experimental contact probability profiles for the mitotic chromosomes in HeLa and DT40 cells. The LE rate is smaller at the start of mitosis and increases as the cells approach metaphase. Condensin II-mediated mean loop size is about six times larger than loops because of condensin I. The loops, which overlap each other, are stapled to a central dynamically changing helical scaffold formed by the motors during the LE process. A polymer physics-based data-driven method that uses the Hi-C contact map as the only input shows that the helix is characterized as random helix perversions (RHPs) in which the handedness changes randomly along the scaffold. The theoretical predictions, which are testable using imaging experiments, do not contain any parameters.

Conformational Fluctuations and Phases in Fused in Sarcoma (FUS) Low-Complexity Domain.

The well known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA binding protein, Fused In Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C- terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like behavior) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site specific and affects the stability of the fibril depending on the sites that are phosphorylated. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular understanding.

Energy landscapes of Aß monomers are sculpted in accordance with Ostwald's rule of stages.

The transition from a disordered to an assembly-competent monomeric state (N*) in amyloidogenic sequences is a crucial event in the aggregation cascade. Using a well-calibrated model for intrinsically disordered proteins (IDPs), we show that the N* states, which bear considerable resemblance to the polymorphic fibril structures found in experiments, not only appear as excitations in the free energy landscapes of Aß40 and Aß42, but also initiate the aggregation cascade. For Aß42, the transitions to the different N* states are in accord with Ostwald's rule of stages, with the least stable structures forming ahead of thermodynamically favored ones. The Aß40 and Aß42 monomer landscapes exhibit different extents of local frustration, which we show have profound implications in dictating subsequent self-assembly. Using kinetic transition networks, we illustrate that the most favored dimerization routes proceed via N* states. We argue that Ostwald's rule also holds for the aggregation of fused in sarcoma and polyglutamine proteins.

A maximum-entropy model to predict 3D structural ensembles of chromatin from pairwise distances with applications to interphase chromosomes and structural variants.

The principles that govern the organization of genomes, which are needed for an understanding of how chromosomes are packaged and function in eukaryotic cells, could be deciphered if the three-dimensional (3D) structures are known. Recently, single-cell imaging techniques have been developed to determine the 3D coordinates of genomic loci in vivo. Here, we introduce a computational method (Distance Matrix to Ensemble of Structures, DIMES), based on the maximum entropy principle, with experimental pairwise distances between loci as constraints, to generate a unique ensemble of 3D chromatin structures. Using the ensemble of structures, we quantitatively account for the distribution of pairwise distances, three-body co-localization, and higher-order interactions. The DIMES method can be applied to both small and chromosome-scale imaging data to quantify the extent of heterogeneity and fluctuations in the shapes across various length scales. We develop a perturbation method in conjunction with DIMES to predict the changes in 3D structures from structural variations. Our method also reveals quantitative differences between the 3D structures inferred from Hi-C and those measured in imaging experiments. Finally, the physical interpretation of the parameters extracted from DIMES provides insights into the origin of phase separation between euchromatin and heterochromatin domains.