Toward Accurate Simulation of Coupling between Protein Secondary Structure and Phase Separation.

Intrinsically disordered proteins (IDPs) frequently mediate phase separation that underlies the formation of a biomolecular condensate. Together with theory and experiment, efficient coarse-grained (CG) simulations have been instrumental in understanding the sequence-specific phase separation of IDPs. However, the widely used Cα-only models are limited in capturing the peptide nature of IDPs, particularly backbone-mediated interactions and effects of secondary structures, in phase separation. Here, we describe a hybrid resolution (HyRes) protein model toward a more accurate description of the backbone and transient secondary structures in phase separation. With an atomistic backbone and coarse-grained side chains, HyRes can semiquantitatively capture the residue helical propensity and overall chain dimension of monomeric IDPs. Using GY-23 as a model system, we show that HyRes is efficient enough for the direct simulation of spontaneous phase separation and, at the same time, appears accurate enough to resolve the effects of single His to Lys mutations. HyRes simulations also successfully predict increased ß-structure formation in the condensate, consistent with available experimental CD data. We further utilize HyRes to study the phase separation of TPD-43, where several disease-related mutants in the conserved region (CR) have been shown to affect residual helicities and modulate the phase separation propensity as measured by the saturation concentration. The simulations successfully recapitulate the effect of these mutants on the helicity and phase separation propensity of TDP-43 CR. Analyses reveal that the balance between backbone and side chain-mediated interactions, but not helicity itself, actually determines phase separation propensity. These results support that HyRes represents an effective protein model for molecular simulation of IDP phase separation and will help to elucidate the coupling between transient secondary structures and phase separation.

Backbone interactions and secondary structures in phase separation of disordered proteins.

Intrinsically disordered proteins (IDPs) are one of the major drivers behind the formation and characteristics of biomolecular condensates. Due to their inherent flexibility, the backbones of IDPs are significantly exposed, rendering them highly influential and susceptible to biomolecular phase separation. In densely packed condensates, exposed backbones have a heightened capacity to interact with neighboring protein chains, which might lead to strong coupling between the secondary structures and phase separation and further modulate the subsequent transitions of the condensates, such as aging and fibrillization. In this mini-review, we provide an overview of backbone-mediated interactions and secondary structures within biomolecular condensates to underscore the importance of protein backbones in phase separation. We further focus on recent advances in experimental techniques and molecular dynamics simulation methods for probing and exploring the roles of backbone interactions and secondary structures in biomolecular phase separation involving IDPs.

Polymers for Disrupting Protein-Protein Interactions: Where Are We and Where Should We Be?

Protein-protein interactions (PPIs) are central to the cellular signaling and regulatory networks that underlie many physiological and pathophysiological processes. It is challenging to target PPIs using traditional small molecule or peptide-based approaches due to the frequent lack of well-defined binding pockets at the large and flat PPI interfaces. Synthetic polymers offer an opportunity to circumvent these challenges by providing unparalleled flexibility in tuning their physiochemical properties to achieve the desired binding properties. In this review, we summarize the current state of the field pertaining to polymer-protein interactions in solution, highlighting various polyelectrolyte systems, their tunable parameters, and their characterization. We provide an outlook on how these architectures can be improved by incorporating sequence control, foldability, and machine learning to mimic proteins at every structural level. Advances in these directions will enable the design of more specific protein-binding polymers and provide an effective strategy for targeting dynamic proteins, such as intrinsically disordered proteins.

One-Dimensional Helical Nanostructures from the Hierarchical Self-Assembly of an Achiral "Rod-Coil" Alternating Copolymer.

The self-assembly of alternating copolymers (ACPs) has attracted considerable interest due to their unique alternating nature. However, compared with block copolymers, their self-assembly behavior remains much less explored and their reported self-assembled structures are limited. Here, the formation of supramolecular helical structures by the self-assembly of an achiral rod-coil alternating copolymer named as poly(quarter(3-hexylthiophene)-alt-poly(ethylene glycol)) (P(Q3HT-alt-PEG)), is reported. The copolymer exhibits an interesting hierarchical self-assembly process, driven by the π-π stacking of the Q3HT segments and the solvophobic interaction of the alkyl chains in tetrahydrofuran (THF)-isopropanol mixed solvents. The copolymer first self-assembled into thin nanobelts with a uniform size, then grows to helical nanoribbons and eventually twisted into helical nanowires with an average diameter of 25 ± 9 nm and a mean pitch of 80 ± 10 nm. Dissipative particle dynamics (DPD) simulation supports the formation course of the helical nanowires. Furthermore, the addition of (S)-ethyl lactate and (R)-ethyl lactate in the self-assembly of P(Q3HT-alt-PEG) results in the formation of left-handed and right-handed chiral nanowires, respectively, demonstrating the tunability of the chirality of the helical wires. This study expands the library of ordered self-assembled structures of ACPs, and also brings a new strategy and mechanism to construct helical supramolecular structures.

Membrane-Bound Inward-Growth of Artificial Cytoskeletons and Their Selective Disassembly.

The cytoskeleton is one of the most important cellular components. Up to now, most of the reported artificial cytoskeletons are based on a gel-in-vesicle strategy. Herein, we report a membrane-bound inward-growth pathway to prepare cytoskeleton-like and radially aligned nanofibers grown from capsule membranes to get membrane-bound artificial cytoskeletons (MACs). The mechanism therein is disclosed through the direct observation of the intermediates in both dried and liquid states. Furthermore, the as-prepared MACs show a selective disassembly behavior in the presence of reductants: both capsule membranes and MACs can be disassembled or only MACs can be disassembled through the selective introduction of dynamic disulfide bonds (DS) into them and by the switch of ultraviolet (UV) irradiation. The present work provides a new hierarchical self-assembly way to construct artificial cytoskeletons with controlled compositions and orientations.

Self-Assembly of Peapod-like Micrometer Tubes from a Planet-Satellite-type Supramolecular Megamer.

This study presents interesting self-assembly of peapod-like micrometer tubes from a planet-satellite-type supramolecular megamer, which was constructed through the specific host-guest molecular recognition between azobenzene (AZO)-functionalized hyperbranched poly(ethyl-3-oxetanemethanol)-star-poly(ethylene oxide) (HSP-AZO) and ß-cyclodextrin(CD)-based hydrophilic hyperbranched polyglycerol (CD-g-HPG). A peapod-like structure with micrometer-sized tube as the pod and vesicles encapsulated inside as the peas was formed through sequential vesicle entosis, linear association, and fusion processes. Dissipative particle dynamics (DPD) simulations support the structural possibility of the supramolecular peapod formation and its mechanism. UV light irradiation could lead to the disassembly of the peapod-like structure. This study expands the family of supramolecular polymers and opens a new avenue to develop bioinspired complex hierarchical nanoarchitectures at the microscopic level.

Dynamics and multi-annual fate of atmospherically deposited nitrogen in montane tropical forests.

The effects of nitrogen (N) deposition on forests largely depend on its fate after entering the ecosystem. While several studies have addressed the forest fate of N deposition using 15 N tracers, the long-term fate and redistribution of deposited N in tropical forests remains unknown. Here, we applied 15 N tracers to examine the fates of deposited ammonium ( NH 4 + ) and nitrate ( NO 3 - ) separately over 3 years in a primary and a secondary tropical montane forest in southern China. Three months after 15 N tracer addition, over 60% of 15 N was retained in the forests studied. Total ecosystem retention did not change over the study period, but between 3 months and 3 years following deposition 15 N recovery in plants increased from 10% to 19% and 13% to 22% in the primary and secondary forests, respectively, while 15 N recovery in the organic soil declined from 16% to 2% and 9% to 2%. Mineral soil retained 50% and 35% of 15 N in the primary and secondary forests, with retention being stable over time. The total ecosystem retention of the two N forms did not differ significantly, but plants retained more 15 NO 3 - than 15 NH 4 + and the organic soil more 15 NH 4 + than NO 3 - . Mineral soil did not differ in 15 NH 4 + and 15 NO 3 - retention. Compared to temperate forests, proportionally more 15 N was distributed to mineral soil and plants in these tropical forests. Overall, our results suggest that atmospherically deposited NH 4 + and NO 3 - is rapidly lost in the short term (months) but thereafter securely retained within the ecosystem, with retained N becoming redistributed to plants and mineral soil from the organic soil. This long-term N retention may benefit tropical montane forest growth and enhance ecosystem carbon sequestration.

pH-Controlled Stereoregular Polymerization of Poly(methyl methacrylate) in Vesicle Membranes.

Here, we report a pH-controlled stereoregular polymerization of methyl methacrylate (MMA) inside the membrane of H20-COOH hyperbranched polymer vesicles using a common radical polymerization process. The vesicle size decreases from 745 to 214 nm with an increase of solution pH from 2.60 to 7.26, and the isotacticity of the obtained polymethyl methacrylates (PMMAs) is accordingly elevated from 9 to 35%. The obtained isotactic-rich PMMAs show a lower glass transition temperature depending on the isotacticity than the commercial random PMMAs. A mechanism study according to the in situ Fourier transform infrared measurements indicates that the control of polymer isotacticity results from the monomer conformation confined effect inside the thin vesicle membranes. The present study provides a new method to realize the preparation of isotactic polymers with the characteristics of facile synthesis, pH controllability, and a green polymerization process in aqueous solution as well as under mild reaction conditions of ambient temperature and pressure.

Computational design of Janus polymersomes with controllable fission from double emulsions.

Janus polymer vesicles (polymersomes) with biphasic membranes have special properties and potential applications in many fields. The big barrier for the preparation of Janus polymersomes lies in the difficulty of complete lateral microphase separation of polymers along the vesicle membrane due to the limited mobility. Herein, we present a systematic simulation study to provide a new strategy for the fabrication of Janus polymersomes based on water-in-oil-in-water double emulsions. Two incompatible block copolymers of AB and AC completely separate into two hemispheres of the polymersome driven by the dewetting of double emulsions, followed by the stabilization of the Janus structure with the block copolymers BC at the interface between AB and AC hemispheres. The simulation results demonstrate the formation of Janus polymersomes in a wide range of the incompatibility between blocks B and C. In addition, the morphologies of the Janus polymersomes can be readily regulated by changing the number of copolymers BC, the ratio of AB to AC, and the dewetting rate of organic solvents. Both the Janus and patchy polymersomes can be obtained through the adjustment of the dewetting rate. Besides, by introducing stimulus-cleavable copolymers of BC, the Janus polymersomes can perform controllable fission. Further comparison with similar experiments has also demonstrated the feasibility of our strategy. We believe the present work will be useful for the fabrication of polymersomes with controlled patches in a large quantity, and the stimulus-responsive fission process will also make the polymersomes promising in some applications like controlled drug delivery and cytomimetic membrane communication.

High-χ alternating copolymers for accessing sub-5 nm domains via simulations.

The ever-growing semiconductor industry has encouraged the feature dimensions of nanolithography to reach the sub-10 nm length scale. It is highly necessary to find nanolithographic materials with high performance but ultra-small domains. We have designed a series of high-χ alternating copolymers (ACPs), in which the polar and apolar repeating units are four hydroxyl groups and alkyl chains, respectively. Careful coarse-grained molecular dynamics (CG-MD) simulations demonstrate that these ACPs can form a variety of mesophases, including lamellae, perforated lamellae, and hexagonally packed cylinders. All the domain periods of these mesophases are smaller than 5 nm, and the smallest domain is close to 1 nm. Most importantly, both the phase morphologies and domain periods are independent of the molecular weight (MW) and molecular weight distribution (MWD) when the degree of polymerization (N) exceeds the threshold value. Thus, using high-χ ACPs, ultranarrow domains can be realized with high MW for sufficient material performance, while the MWD-independence can ensure the uniformity of the domain sizes. We believe that these "high χ-high N" alternating copolymers are promising alternatives as new nanolithographic materials.

In silico study of structure and water dynamics in CNT/polyamide nanocomposite reverse osmosis membranes.

CNT-based reverse osmosis membranes have long been regarded as one of the most promising candidates for water desalination. However, it is a pity that there is no complete understanding of the exact role of CNTs in those nanocomposite membranes. To address this issue, three atomistic models of PA (pure polyamide membrane), PA-CNT1 (polyamide nanocomposite membrane with an embedded carbon nanotube oriented vertical to the membrane surface) and PA-CNT2 (polyamide nanocomposite with an embedded carbon nanotube oriented parallel to the membrane surface) were constructed respectively in this work. Then, equilibrium molecular dynamics (EMD) and non-equilibrium molecular dynamics (NEMD) simulations were conducted to investigate the structure and water dynamics in these three models. The EMD simulations revealed a better stacking of the PA matrix due to the addition of the CNT and this impact was more significant in PA-CNT1 than in PA-CNT2. Meanwhile, PA matrix near the mouth of the CNT was found to behave as an obstruction that hindered the exchange of water molecules inside and outside the CNT. In NEMD simulations, we found that water molecules were guided away from the CNT because of the better stacked surrounding PA matrix. The partially covered CNT might not help to increase water flux in PA-CNT1 while guided water molecules and the smaller polymer region afftected by the CNT contributed to a relatively high flux in PA-CNT2. The current work might serve as a comprehensive understanding of the role of CNTs in the reverse osmosis process.

MembrFactory: A Force Field and composition Double Independent Universal Tool for Constructing Polyamide Reverse Osmosis Membranes.

The topmost polyamide (PA) layer of the thin-film-composite reverse osmosis (RO) membrane is the most important part in the membrane-based RO technology. With the aid of molecular dynamics simulations, many PA layer-related features in the RO process can be revealed. With many novel types of PA RO membranes out of trimesoyl chloride/m-phenylenediamine monomers developed in the laboratory, a convenient model building tool for these PA layer systems is urgently needed to conduct the theoretical analysis. Here, we develop a new universal toolkit for constructing PA RO membranes, named as MembrFactory, which combines flexibility of force fields and membrane compositions. A key characteristic of our approach is the use of monomers as the starting state, and the final membrane model was obtained automatically by stepwise reaction between the functional groups on the monomers. The reliability of MembrFactory has been validated by constructing several common PA RO membranes. © 2019 Wiley Periodicals, Inc.

Self-assembly of Amphiphilic Alternating Copolymers.

Polymer self-assembly has been a hot research topic for several decades. Different types of polymers with various architectures, like block copolymers, brush polymers, hyperbranched polymers and dendrimers, etc., are currently being investigated. Alternating copolymers (ACPs) are regular copolymers with an alternating monomeric unit structure in the polymer backbones. However, despite the great progress in the synthesis of ACPs, their self-assembly is still in an infant stage. Very recently, our group reported a new type of amphiphilic ACPs through click copolymerization and obtained spheres, vesicles, nanotubes, and even hierarchical sea urchin-like aggregates through the self-assembly process. In addition, we have found some intriguing features in the self-assembly of amphiphilic ACPs when compared with other copolymers, including their facile syntheses, readily functionalization, novel self-assembly structures, new folding-chain mechanisms, and uniform but ultrathin feature length. In this Concept article, we present the self-assembly of amphiphilic ACPs together with their unique features by reviewing our latest results and related studies. Moreover, the future perspective on the self-assembly of amphiphilic ACPs is also proposed. Our aim is to capture the attention and interest of chemists in this new area of polymerization.

Shape Transformations of Vesicles Self-Assembled from Amphiphilic Hyperbranched Multiarm Copolymers via Simulation.

The understanding of shape transformations of vesicles is of fundamental importance in biological and clinical sciences. Hyperbranched polymer vesicles (branched polymersomes) are newly emerging polymer vesicles with special structure and property. They have also been regarded as a good model for biomembranes. However, the shape transformations of hyperbranched polymer vesicles have not been studied from either an experimental or theoretical level. Herein, the shape transformations of vesicles self-assembled from amphiphilic hyperbranched multiarm copolymers (HMCs) in response to the interaction parameters between the hydrophobic core and hydrophilic arms and the polymer concentrations are investigated carefully through dissipative particle dynamics (DPD) simulations. In the morphological phase diagram, two types of vesicles are obtained: one type corresponds to vesicles without holes formed at low concentrations including unilamellar vesicles, double-lamellar vesicles, discocyte-shaped vesicles, and tubular vesicles, and the other type corresponds to vesicles with holes formed at high concentrations including stomatocyte-shaped vesicles, toroidal vesicles, genus-3 (G-3) toroidal vesicles with three holes, and genus-4 (G-4) toroidal vesicles with four holes. In addition, both the self-assembly mechanisms and the dynamics for the formation of these vesicles have been systematically studied. The current work will offer theoretical support for fabricating novel vesicles with various shapes from hyperbranched polymers.

Fate of atmospherically deposited NH4+ and NO3- in two temperate forests in China: temporal pattern and redistribution.

The impacts of anthropogenic nitrogen (N) deposition on forest ecosystems depend in large part on its fate. However, our understanding of the fates of different forms of deposited N as well as the redistribution over time within different ecosystems is limited. In this study, we used the 15 N-tracer method to investigate both the short-term (1 week to 3 months) and long-term (1-3 yr) fates of deposited NH4+ or NO3- by following the recovery of the 15 N in different ecosystem compartments in a larch plantation forest and a mixed forest located in northeastern China. The results showed similar total ecosystem retention for deposited NH4+ and NO3- , but their distribution within the ecosystems (plants vs. soil) differed distinctly particularly in the short-term, with higher 15 NO3- recoveries in plants (while lower recoveries in organic layer) than found for 15 NH4+ . The different short-term fate was likely related to the higher mobility of 15 NO3- than 15 NH4+ in soils instead of plant uptake preferences for NO3- over NH4+ . In the long-term, differences between N forms became less prevalent but higher recoveries in trees (particularly in the larch forest) of 15 NO3- than 15 NH4+ tracer persisted, suggesting that incoming NO3- may contribute more to plant biomass increment and forest carbon sequestration than incoming NH4+ . Differences between the two forests in recoveries were largely driven by a higher 15 N recovery in the organic layer (both N forms) and in trees (for 15 NO3- ) in the larch forest compared to the mixed forest. This was due to a more abundant organic layer and possibly higher tree N demand in the larch forest than in the mixed forest. Leachate 15 N loss was minor (<1% of the added 15 N) for both N forms and in both forests. Total 15 N recovery averaged 78% in the short-term and decreased to 55% in the long-term but with increasing amount of 15 N label (re)-redistributed into slow turn-over pools (e.g., trees and mineral soil). The different retention dynamics of deposited NH4+ and NO3- may have implications in environmental policy related to the anthropogenic emissions of the two N forms.

Solution self-assembly behavior of rod-alt-coil alternating copolymers via simulations.

Alternating copolymers (ACPs) have shown several attractive unique characteristics in solution self-assembly due to their special alternating structure. With the introduction of rod segments, much more complexity and multifunctionality can be achieved in the self-assembly of rod-alt-coil ACPs. Herein, we have performed a simulation study on the self-assembly of rod-alt-coil ACPs in dilute solution through dissipative particle dynamics (DPD) simulations. A morphological phase diagram was constructed as a function of rod and coil length, in which diverse assemblies were found, such as bicontinuous micelles and perforated membranes. Furthermore, the alignment of rod segments in the assemblies has been disclosed in detail. And, we deeply investigated the effects of rod length, coil length and π-π interaction strength on the self-assembly morphologies and rod alignment. With the increase of rod length, a disorder-order transition was observed, and π-π interactions could facilitate the orderly alignment of rods. Besides, our simulation results showed good agreement with available experiments. Furthermore, the unique characteristics in the self-assembly of rod-alt-coil alternating copolymers were discussed; in particular we found that the alternating molecular structures of ACPs could promote the orderly alignment of rod segments. We believe that the current work can provide a solid theoretical foundation for further experimental studies.

Molecular dynamics simulation studies of the structure and antifouling performance of a gradient polyamide membrane.

The polyamide (PA) layer on the surface of thin-film-composite reverse osmosis membranes is the core aspect of membrane-based desalination technology. In recent years, molecular dynamics simulations have been increasingly used to disclose the physicochemical properties of the PA layer. However, the currently reported all-atom PA layer models do not exhibit gradient variation of the structural properties of the layer, and they can only represent the innermost region of the PA layer. With the help of our recently developed universal toolkit "MembrFactory", this paper reports a modeling method that can be used to construct a gradient crosslinking model and surface grafting model for the PA layer. A fully atomistic model of the PA layer was constructed, in which the degree of crosslinking (DC) was changed gradiently along the thickness direction. The structure of the PA layer model and the transport dynamics of the water molecules within it were systematically investigated using equilibrium molecular dynamics simulations. We found that the DC is the lowest and the water molecules have the strongest self-diffusion ability in the interfacial region of the PA layer model. Meanwhile, the pore size is distributed widely in the region. Subsequently, we modified the surface of the PA layer model with PEG coatings, and their coverage ratio was around 75%. The radial distribution function analysis showed that water molecules prefer to coordinate with the oxygen atoms in PEG. Furthermore, two contaminant molecules, 1-ethyl-2-methyl benzene and n-decane, were selected to investigate the antifouling properties of the PEG-modified PA layer. By analysing the trajectories of the pollutants and calculating the potential of the mean force, we found that the antifouling performance of a PEG-modified PA layer is not only related to the hydrophobicity and the size of the pollutant, but is also related to the coverage ratio of the PEG layer.

Understanding the temperature effect on transport dynamics and structures in polyamide reverse osmosis system via molecular dynamics simulations.

The structures and transport dynamics of water and salt ions in polyamide (PA) reverse osmosis (RO) membranes as well as the temperature effects on the RO process were systematically investigated using a fully atomistic simulation method. By comparing the experimental data of the commercial membrane FT-30 and the available MD simulation results, the reliability of our PA RO model was validated. In addition, the groups on the polymer chains that preferentially participated in the coordination shells of salt ions were determined. Moreover, we found that the self-diffusion coefficients of both ions reduced by two orders of magnitude due to interactions between the ions and the polymer chains. Furthermore, NEMD simulations showed that the temperature has both positive and negative effects on the water flux. Although increasing the temperature can enhance the mobility of water molecule, it also can reduce the size of water clusters, which hampers an increase in the water flux. The decrease in size of the largest water clusters can partly explain the decrease in water flux when salt ions exist in the membrane. The current work provides a comprehensive understanding of the structure and transport behaviour of water and salt ions in the RO membranes.

Emulsion-Assisted Polymerization-Induced Hierarchical Self-Assembly of Giant Sea Urchin-like Aggregates on a Large Scale.

Hierarchical solution self-assembly has become an important biomimetic method to prepare highly complex and multifunctional supramolecular structures. However, despite great progress, it is still highly challenging to prepare hierarchical self-assemblies on a large scale because the self-assembly processes are generally performed at high dilution. Now, an emulsion-assisted polymerization-induced self-assembly (EAPISA) method with the advantages of in situ self-assembly, scalable preparation, and facile functionalization was used to prepare hierarchical multiscale sea urchin-like aggregates (SUAs). The obtained SUAs from amphiphilic alternating copolymers have a micrometer-sized rattan ball-like capsule (RBC) acting as the hollow core body and radiating nanotubes tens of micrometers in length as the hollow spines. They can capture model proteins effectively at an ultra-low concentration (ca. 10 nm) after functionalization with amino groups through click copolymerization.

Asymmetric Polymersomes from an Oil-in-Oil Emulsion: A Computer Simulation Study.

Asymmetric vesicles are valuable for understanding and mimicking cell and practical biomedicine applications. Recently, a very straightforward methodology for fabricating asymmetric polymersome was developed by Lodge's group through the coassembly of polystyrene-b-poly(ethylene oxide) (PS-b-PEO) and polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) block copolymers at the interface of a polystyrene/polybutadiene/chloroform (PS/PB/CHCl3) emulsion. However, the in-depth microscopic mechanism for the formation of asymmetric polymersomes remains unclear. To address this issue, in this article, the coassembly process for the formation of the asymmetric polymersomes in Asano's experimental system was systematically investigated by employing a dissipative particle dynamics (DPD) simulation. Our results definitely demonstrate the formation of the asymmetric polymersomes such as that in the experiments and that the bilayer formed through the folding and crossing of the PEO blocks. Besides, from the microscopic view, three stages can be discerned in the formation process: (1) the formation of micelles, (2) the micelle diffusion to the interface, and (3) the micelle rearrangement at the interface to form an asymmetric polymersome. Meanwhile, the incompatibility among PS, PB, and PEO is proven to be the main driving force for asymmetric polymersome formation. Moreover, the effects of the order of addition of copolymers and the volume fraction of PEO blocks on the structure of the asymmetric polymersomes are also investigated. We find that the formation process is affected severely by the order of addition, and adding PS-b-PEO first can make the asymmetric bilayer more perfect. Not only that, but perfect asymmetric polymersomes can be formed only when the volume fraction of PEO (fPEO) is greater than 0.55. We believe the present work can extend the knowledge of the self-assembly of asymmetric polymersomes, especially with respect to the self-assembly mechanism.