Random Heteropolymers Research Articles

Sequence‐sensitivity in functional synthetic polymer properties

Recently, a new class of synthetic methyl methacrylate‐based random heteropolymers (MMA‐based RHPs) has displayed protein‐like properties. Their function appears to be insensitive to the precise sequence. Here, through atomistic molecular dynamics simulation, we show that there are universal protein‐like features of MMA‐based RHPs that are insensitive to the sequence, and mostly depend on the overall composition. In particular, we find that MMA‐based RHPs ``fold'' into globules with heterogeneous hydration patterns. However, the insensitivity to sequence identity observed in MMA‐based RHPs dramatically changes when we substitute the backbone architecture with acrylate or replace the oxygen atom in the side chain with a nitrogen atom (methacrylamide or acrylamide). Using principal component analysis and an intersection‐over‐union based index, we demonstrate that different sequences may not overlap in the property space, meaning that their properties are controlled by the sequence rather than fixed composition. We further investigate the sequence‐insensitive capability of the MMA‐based RHPs as previously reported on bacterial phospholipase OmpLA stabilization through heterodimerization. As experimentally observed, such polymers enhance the stability of OmpLA as reliably as its native bilayer environment. The design of such MMA‐based RHPs provides a sequence‐insensitive alternative to protein‐mimetic biomaterials that is orthogonal to the sequence‐structure‐function paradigm of proteins.

Sequence-sensitivity in functional synthetic polymer properties.

Recently, a new class of synthetic methyl methacrylate-based random heteropolymers (MMA-based RHPs) has displayed protein-like properties. Their function appears to be insensitive to the precise sequence. Here, through atomistic molecular dynamics simulation, we show that there are universal protein-like features of MMA-based RHPs that are insensitive to the sequence, and mostly depend on the overall composition. In particular, we find that MMA-based RHPs ``fold'' into globules with heterogeneous hydration patterns. However, the insensitivity to sequence identity observed in MMA-based RHPs dramatically changes when we substitute the backbone architecture with acrylate or replace the oxygen atom in the side chain with a nitrogen atom (methacrylamide or acrylamide). Using principal component analysis and an intersection-over-union based index, we demonstrate that different sequences may not overlap in the property space, meaning that their properties are controlled by the sequence rather than fixed composition. We further investigate the sequence-insensitive capability of the MMA-based RHPs as previously reported on bacterial phospholipase OmpLA stabilization through heterodimerization. As experimentally observed, such polymers enhance the stability of OmpLA as reliably as its native bilayer environment. The design of such MMA-based RHPs provides a sequence-insensitive alternative to protein-mimetic biomaterials that is orthogonal to the sequence-structure-function paradigm of proteins.

Mapping Composition Evolution through Synthesis, Purification, and Depolymerization of Random Heteropolymers.

Random heteropolymers (RHPs) consisting of three or more comonomers have been routinely used to synthesize functional materials. While increasing the monomer variety diversifies the side-chain chemistry, this substantially expands the sequence space and leads to ensemble-level sequence heterogeneity. Most studies have relied on monomer composition and simulated sequences to design RHPs, but the questions remain unanswered regarding heterogeneities within each RHP ensemble and how closely these simulated sequences reflect the experimental outcomes. Here, we quantitatively mapped out the evolution of monomer compositions in four-monomer-based RHPs throughout a design-synthesis-purification-depolymerization process. By adopting a Jaacks method, we first determined 12 reactivity ratios directly from quaternary methacrylate RAFT copolymerization experiments to account for the influences of competitive monomer addition and the reversible activation/deactivation equilibria. The reliability of in silico analysis was affirmed by a quantitative agreement (<4% difference) between the simulated RHP compositions and the experimental results. Furthermore, we mapped out the conformation distribution within each ensemble in different solvents as a function of monomer chemistry, composition, and segmental characteristics via high-throughput computation based on self-consistent field theory (SCFT). These comprehensive studies confirmed monomer composition as a viable design parameter to engineer RHP-based functional materials as long as the reactivity ratios are accurately determined and the livingness of RHP synthesis is ensured.

Molecular Motor-Driven Light-Controlled Logic-Gated K+ Channel for Cancer Cell Apoptosis.

Developing artificial ion transport systems, which process complicated information and step-wise regulate properties, is essential for deeply comprehending the subtle dynamic behaviors of natural channel proteins (NCPs). Here a photo-controlled logic-gated K+ channel based on single-chain random heteropolymers containing molecular motors, exhibiting multi-core processor-like properties to step-wise control ion transport is reported. Designed with oxygen, deoxygenation, and different wavelengths of light as input signals, complicated logical circuits comprising "YES", "AND", "OR" and "NOT" gate components are established. Implementing these logical circuits with K+ transport efficiencies as output signals, multiple state transitions including "ON", "Partially OFF" and "Totally OFF" in liposomes and cancer cells are realized, further causing step-wise anticancer treatments. Dramatic K+ efflux in the "ON" state (decrease by 50% within 7min) significantly induces cancer cell apoptosis. This integrated logic-gated strategy will be expanded toward understanding the delicate mechanism underlying NCPs and treating cancer or other diseases is expected.

Sequence-dependent and -independent information in a combined random energy model for protein folding and coding.

Random energy models (REMs) provide a simple description of the energy landscapes that guide protein folding and evolution. The requirement of a large energy gap between the native structure and unfolded conformations, considered necessary for cooperative, protein-like, folding behavior, indicates that proteins differ markedly from random heteropolymers. It has been suggested, therefore, that natural selection might have acted to choose nonrandom amino acid sequences satisfying this particular condition, implying that a large fraction of possible, unselected random sequences, would not fold to any structure. From an informational perspective, however, this scenario could indicate that protein structures, regarded as messages to be transmitted through a communication channel, would not be efficiently encoded in amino acid sequences, regarded as the communication channel for this transmission, since a large fraction of possible channel states would not be used. Here, we use a combined REM for conformations and sequences, with previously estimated parameters for natural proteins, to explore an alternative possibility in which the appropriate shape of the landscape results mainly from the deviation from randomness of possible native structures instead of sequences. We observe that this situation emerges naturally if the distribution of conformational energies happens to arise from two independent contributions corresponding to sequence-dependent and -independent terms. This construction is consistent with the hypothesis of a protein burial folding code, with native structures being determined by a modest amount of sequence-dependent atomic burial information with sequence-independent constraints imposed by unspecific hydrogen bond formation. More generally, an appropriate combination of sequence-dependent and -independent information accommodates the possibility of an efficient structural encoding with the main physical requirement for folding, providing possible insight not only on the folding process but also on several aspects sequence evolution such as neutral networks, conformational coverage, and de novo gene emergence.

Dynamic transformation of bio-inspired single-chain nanoparticles at interfaces.

The interfacial behavior of macromolecules dictates their intermolecular interactions, which can impact the processing and application of polymers for pharmaceutical and synthetic use. Using molecular dynamics simulations, we observe the evolution of a random heteropolymer in the presence of liquid-liquid interfaces. The system of interest forms single-chain nanoparticles through hydrophobic collapse in water, lacking permanent crosslinks and making their morphology mutable in new environments. Complex amphiphilic polymers are shown to be capable of stabilizing high interfacial tension water-hexane interfaces, often unfolding to maximize surface coverage. Despite drastic changes to polymer conformation, monomer presence in the water phase is generally maintained and most changes are due to increased hydrophobic solvent exposure toward the oil phase. These results are then compared to the behavior at the water-graphene interface, where the macromolecules adsorb but do not remodel. The polymer's behavior is shown to depend significantly on both its own amphiphilic character and the deformability of the interface.

Ensembles of synthetic polymers mimic biological fluids

Recently a report by Ruan et al. in Nature described how relatively simple random heteropolymers can replicate the properties of biological fluids. These polymers capture the segmental-level interactions between proteins and could enhance folding of membrane proteins, improve stability, and enable DNA sequestration in a chemistry specific manner.

Adsorption of Biomimetic Amphiphilic Heteropolymers onto Graphene and Its Derivatives

The adsorption of a polymer chain from a bulk solution onto a solid surface is the fundamental mechanism behind many industrial and biological applications. Here, we used molecular dynamics simulations to investigate the driving factors in the adsorption of a poly(methyl methacrylate)-based biomimetic random heteropolymer (RHP) containing polar (ethylene glycol, or PEG), nonpolar (ethylhexyl), and charged groups (sulfopropyl acid) onto graphene and its derivatives (graphene oxide and carbon nanotube). We applied a combination of unbiased and biased simulations with machine learning to investigate the adsorption mechanism. The long PEG side chains drive the nearby short monomers to be adsorbed. After a short period of side-chain adsorption, due to the glassy nature of the backbone, the adsorption terminates and will only proceed by adding plasticizers, changing the backbone to be more flexible, or raising the temperature over the glass transition temperature. Meanwhile, thermodynamically, the adsorption is favorable due to the amphiphilicity of PEG. The surface hydrophilicity and curvature influence the adsorption as well. This work provides atomistic details of the interaction between an amphiphilic RHP and a hard surface, which can serve as the basis to understand the adsorption of such RHPs on more complex interfaces such as protein surfaces.

Sequence Design of Random Heteropolymers as Protein Mimics.

Random heteropolymers (RHPs) have been computationally designed and experimentally shown to recapitulate protein-like phase behavior and function. However, unlike proteins, RHP sequences are only statistically defined and cannot be sequenced. Recent developments in reversible-deactivation radical polymerization allowed simulated polymer sequences based on the well-established Mayo-Lewis equation to more accurately reflect ground-truth sequences that are experimentally synthesized. This led to opportunities to perform bioinformatics-inspired analysis on simulated sequences to guide the design, synthesis, and interpretation of RHPs. We compared batches on the order of 10000 simulated RHP sequences that vary by synthetically controllable and measurable RHP characteristics such as chemical heterogeneity and average degree of polymerization. Our analysis spans across 3 levels: segments along a single chain, sequences within a batch, and batch-averaged statistics. We discuss simulator fidelity and highlight the importance of robust segment definition. Examples are presented that demonstrate the use of simulated sequence analysis for in-silico iterative design to mimic protein hydrophobic/hydrophilic segment distributions in RHPs and compare RHP and protein sequence segments to explain experimental results of RHPs that mimic protein function. To facilitate the community use of this workflow, the simulator and analysis modules have been made available through an open source toolkit, the RHPapp.

Heuristic Computational Model for Predicting Lignin Solubility in Tailored Organic Solvents

Lignin is a random heteropolymer that has been extensively studied as a renewable source of aromatic precursors for high-value chemicals, biofuels, and bioplastics. A key challenge in lignin valorization is the structural and compositional heterogeneity of lignin feedstocks. Solvent-based approaches are commonly used to fractionate lignin to reduce this heterogeneity, but solvent selection can be challenging due to variability in lignin composition. In this work, we developed computational methods to predict good and poor organic solvents as a function of lignin composition. We analyzed 28 different linear pentamer structures, 18 from known libraries and 10 hypothetical polymers, and calculated their activity coefficients in 50 different organic solvents by using the conductor-like screening model for realistic solvents. We used these data to train a regression model that enabled the extensive investigation of the impact of solvent and monolignol compositions on predicted lignin solubility. The exhaustive exploration of solubility trends using model predictions revealed sets of solvents, identified using Kamlet–Taft parameters, that are predicted to promote lignin dissolution regardless of lignin composition. We further identified solvents expected to selectively isolate lignin fractions enriched in certain subunits. These results establish heuristic guidelines for solvent selection that can be used to tailor fractionation processes for lignin feedstocks of distinct composition or to design new processes that isolate fractions with higher proportions of selected subunits.

Glass transition of random heteropolymers: A molecular dynamics simulation study in melt, in water, and in vacuum

Random heteropolymers (RHPs) have a long history of being studied as toy models for protein folding. Previous computational studies applied statistical mechanics or coarse-grained methods to investigate the RHP phase transition; here, we use atomistic molecular dynamics (MD) simulations to explore the factors affecting the glass transition temperature (Tg) and the mobility of a complex class of RHPs. Our RHPs consist of four methyl methacrylate-based monomers with side chains mimicking different classes of amino acids. We explore the dynamical features in the RHP melts with random sequence mixtures and show that instead of a sharp pseudo-second-order glass transition, as for PMMA, RHPs with low ionic contents show a soft and gradual transition and those with increased content of ionic monomers show a sharp transition. Meanwhile, the configurational entropy of the backbone dihedral angle decreases suddenly in both PMMA and RHPs as a common signature of the glass transition. The dynamical spatial heterogeneity during the glass transition is related to both the chemical structure of the monomers and their positions along the chain. Moreover, chain mobility depends on the content of ionic monomers and types of counterions. The comparison with the single-chain nanoparticles (SCNPs) in water and in vacuum shows “breathing dynamics” with higher mobility. Our work characterizes the physicochemical properties of the RHP melts as a group of emerging bio-inspired polymer materials.

Random Heteropolymer Excipients Improve the Colloidal Stability of a Monoclonal Antibody for Subcutaneous Administration.

Developing stable high concentration monoclonal antibody (mAb) formulations is increasingly important to move toward subcutaneous (SC) administration for better patient experience. Challenges stemming from protein-protein interactions in these crowded solutions, such as colloidal instability, limit the feasibility of some formulations because of concerns of safety, product quality, and/or manufacturability. Herein, we report novel random heteropolymer excipients that improve the colloidal stability of a high concentration mAb formulation for SC administration. A library of polymers was synthesized and screened by a high-throughput, absorbance-based assay. The lead polymers were selected and characterized for their ability to alter the precipitation kinetics of a mAb in physiologically relevant conditions using two model systems. Biophysical testing via surface tension measurements, isothermal titration calorimetry (ITC), microscale thermophoresis (MST), and intrinsic fluorescence quenching indicated that the polymers delayed onset of mAb precipitation from a combination of surfactant behaviour and interactions with the protein to prevent protein-protein interactions leading to colloidal instability. The random heteropolymers described are a new class of excipients that may enable development of SC mAb formulations previously inaccessible to patients.

Topological origin of the protein folding transition.

In this paper, a geometrical and thermodynamical analysis of the global properties of the potential energy landscape of a minimalistic model of a polypeptide is presented. The global geometry of the potential energy landscape is supposed to contain relevant information about the properties of a given sequence of amino acids, that is, to discriminate between a random heteropolymer and a protein. By considering the SH3 and PYP protein-sequences and their randomized versions it turns out that, in addition to the standard signatures of the folding transition-discriminating between protein sequences of amino acids and random heteropolymer sequences-also peculiar geometric signatures of the equipotential hypersurfaces in configuration space can discriminate between proteins and random heteropolymers. Interestingly, these geometric signatures are the "shadows" of deeper topological changes that take place in correspondence with the protein folding transition. The protein folding transition takes place in systems with a small number of degrees of freedom (very far from the Avogadro number) and in the absence of a symmetry-breaking phenomenon. Nevertheless, seen from the deepest level of topology changes of equipotential submanifolds of phase space, the protein folding transition fully qualifies as a phase transition.

Flexible Single‐Chain‐Heteropolymer‐Derived Transmembrane Ion Channels with High K+ Selectivity and Tunable pH‐Gated Characteristics

AbstractA series of single‐chain random heteropolymer (RHP)‐derived artificial ion channels with both high K+ selectivity and controllable pH‐gated behaviors were fabricated by a facile “one‐pot” polymerization method. The benzo‐18‐crown‐6 moieties appended on lateral chains of RHPs can form ion‐permeable nanopores and transport K+ over Na+ through the lipid bilayers. The ion permeation selectivity was significantly enhanced by incorporating a cholesterol group to serve as a membrane anchor. Interestingly, similar to natural gated protein channels, on–off switchable characteristics were also realized by integrating an additional acid‐sensitive alkylamine group into the RHP‐derived channel. The unique design strategies have endowed the RHP‐derived ion channels with facile synthetic procedures, desirable membrane compatibility, high K+ selectivity, and tunable pH‐gated properties. This work provides an entry point for future design of novel functional nanochannels.

Flexible Single-Chain-Heteropolymer-Derived Transmembrane Ion Channels with High K+ Selectivity and Tunable pH-Gated Characteristics.

A series of single-chain random heteropolymer (RHP)-derived artificial ion channels with both high K+ selectivity and controllable pH-gated behaviors were fabricated by a facile "one-pot" polymerization method. The benzo-18-crown-6 moieties appended on lateral chains of RHPs can form ion-permeable nanopores and transport K+ over Na+ through the lipid bilayers. The ion permeation selectivity was significantly enhanced by incorporating a cholesterol group to serve as a membrane anchor. Interestingly, similar to natural gated protein channels, on-off switchable characteristics were also realized by integrating an additional acid-sensitive alkylamine group into the RHP-derived channel. The unique design strategies have endowed the RHP-derived ion channels with facile synthetic procedures, desirable membrane compatibility, high K+ selectivity, and tunable pH-gated properties. This work provides an entry point for future design of novel functional nanochannels.

Solvent Remodeling in Single-Chain Amphiphilic Heteropolymer Systems.

This work demonstrates the remodeling of single-chain nanoparticles (SCNPs) upon a transition to organic solvent through molecular dynamics simulations. Methacrylate-based random heteropolymers (RHPs), assembled via transient noncovalent linkages in water, have shown promise in an assortment of applications that harness their bio-inspired properties. While their molecular behavior has been broadly characterized in aqueous environments, many newer applications include the use of organic solvent rather than bio-mimetic conditions. The polymer assemblies, typically driven by the hydrophobic effect in water, are less well understood in nonaqueous solution. Here, a specific RHP system is examined which forms compact globular morphologies in highly polar or highly nonpolar environments while adopting extended conformations in solvents of intermediate polarity. The pivotal role of electrostatic interactions between charge groups in low dielectric mediums is also observed. Finally, high temperature anneal cycles are compared to room temperature transformations to illuminate barriers to remodeling upon environmentalchanges.

Forced Unfolding of Protein-Inspired Single-Chain Random Heteropolymers

Forced Unfolding of Protein-Inspired Single-Chain Random Heteropolymers

Molecular protectors: Superheroes for nanostructures

Molecular protectors: Superheroes for nanostructures

Automated PET-RAFT Polymerization Towards Pharmaceutical Amorphous Solid Dispersion Development.

In pharmaceutical oral drug delivery development, about 90% of drugs in the pipeline have poor aqueous solubility leading to severe challenges with oral bioavailability and translation to effective and safe drug products. Amorphous solid dispersions (ASDs) have been utilized to enhance the oral bioavailability of poorly soluble active pharmaceutical ingredients (APIs). However, a limited selection of regulatory-approved polymer excipients exists for the development and further understanding of tailor-made ASDs. Thus, a significant need exists to better understand how polymers can be designed to interact with specific API moieties. Here, we demonstrate how an automated combinatorial library approach can be applied to the synthesis and screening of polymer excipients for the model drug probucol. We synthesized a library of 25 random heteropolymers containing one hydrophilic monomer (2-hydroxypropyl acrylate (HPA)) and four hydrophobic monomers at varied incorporation. The performance of ASDs made by a rapid film casting method was evaluated by dissolution using ultra-performance liquid chromatography (UPLC) sampling at various time points. This combinatorial library and rapid screening strategy enabled us to identify a relationship between polymer hydrophobicity, monomer hydrophobic side group geometry, and API dissolution performance. Remarkably, the most effective synthesized polymers displayed slower drug release kinetics compared to industry standard polymer excipients, showing the ability to modulate the drug release profile. Future coupling of high throughput polymer synthesis, high throughput screening (HTS), and quantitative modeling would enable specification of designer polymer excipients for specific API functionalities.

Behavior of Protein-Inspired Synthetic Random Heteropolymers

Random heteropolymers (RHPs) are an interesting class of materials useful in many theories and applications. While previous studies typically focused on simplified RHP systems, here we explore a mo...