Spontaneous Liquid Droplet-to-Gel Transition of Citrulline Polypeptide Complexed with Nucleic Acids.

Inside cells, proteins complex with nucleic acids to form liquid droplets resulting from liquid-liquid phase separation. The presence of mutated proteins can change the state of these liquid droplets to solids or gels, triggering neurodegenerative diseases. The mechanism of the liquid to solid or gel transition is still unclear. Solutions of poly(l-ornithine-co-l-citrulline) (PLOC) copolymers, which exhibit upper critical solution temperature-type behavior, change state upon cooling. In this study, we evaluated the effect of nucleic acids complexed with PLOC on phase changes. In the presence of nucleic acids, such as polyC and polyU, PLOC formed liquid droplets at low temperatures. The droplets dissolved at temperatures above the phase separation temperature. The phase separation temperature depended on the chemical structure of the nucleobase, implying that electrostatic and hydrogen bonding interactions between the nucleic acid and PLOC influenced phase separation. Furthermore, the liquid droplets spontaneously changed to gel-like precipitates due to spontaneous release of nucleic acids from the complex. The rate of the liquid droplet-to-gel transition depended on the magnitude of electrostatic and hydrogen bonding interactions between PLOC and nucleic acid. PLOC complexed with mRNA also underwent a liquid droplet-to-gel transition upon the release of mRNA. This work provides insights into the mechanism of pathogenic transitions of the cellular droplets.

Malachite green-derivatized cationic comb-type copolymer acts as a photoresponsive artificial chaperone.

Molecular chaperones play vital roles in various physiological reactions by regulating the folding and assembly of biomacromolecules. We have demonstrated that cationic comb-type copolymers exhibit chaperone activity for anionic biomolecules including DNA and ionic peptide via the formation of soluble interpolyelectrolyte complexes. The development of smart artificial chaperones that can be spatiotemporally controlled by a remotely guided signal would expand the functions of artificial chaperones. Herein, to enable photocontrol of chaperone activity, a cationic comb-type copolymer bearing malachite green as a photoresponsive unit was designed. We first prepared a series of carboxylic acid derivatives of malachite green identified a derivative that could be quickly and quantitatively converted to the cationic form from the nonionic form by photoirradiation. This derivative was conjugated to the cationic comb-type copolymer, poly(allylamine)-graft-poly(ethylene glycol) through a condensation reaction. Upon photoirradiation, the copolymer bearing 9 mol% malachite green enhanced the membrane disruptive activity of acidic peptide E5 and induced morphological changes in liposomes. This demonstration of photoresponsive activation of chaperoning activity of a copolymer suggests that the installation of carboxyl derivatives of malachite green will impart photoresponsiveness to various materials including biopolymers.

Cationic copolymer and crowding agent have a cooperative effect on a Na+-dependent DNAzyme.

DNAzymes are promising agents for theranostics and biosensors. Sodium dependent DNAzymes have been developed for sensing and imaging of Na+, but these DNAzymes have low catalytic activity. Herein, we demonstrate that a molecular crowded environment containing 10 to 40 wt% PEG enhanced the catalytic activity of a Na+-dependent DNAzyme, EtNa, although dextran did not. The cationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) at 0.03 wt% (0.3 g L-1) enhanced the reaction rate of EtNa by 10-fold, which is similar to the acceleration induced by 15 wt% (150 g L-1) PEG. A cooperative impact of the copolymer and crowding agent was observed: the combination resulted in an impressive 46-fold acceleration effect. Thus, the use of a cationic copolymer and a crowding agent is a promising strategy to improve the activity of Na+-dependent DNAzyme-based nanomachines, biosensors, and theranostics, especially in environments lacking divalent metal ions.

A Cationic Copolymer Enhances Responsiveness and Robustness of DNA Circuits.

Toehold-mediated DNA circuits are extensively employed to construct diverse DNA nanodevices and signal amplifiers. However, operations of these circuits are slow and highly susceptive to molecular noise such as the interference from bystander DNA strands. Herein, this work investigates the effects of a series of cationic copolymers on DNA catalytic hairpin assembly, a representative toehold-mediated DNA circuit. One copolymer, poly(L -lysine)-graft-dextran, significantly enhances the reaction rate by 30-fold due to its electrostatic interaction with DNA. Moreover, the copolymer considerably alleviates the circuit's dependency on the length and GC content of toehold, thereby enhancing the robustness of circuit operation against molecular noise. The general effectiveness of poly(L -lysine)-graft-dextran is demonstrated through kinetic characterization of a DNA AND logic circuit. Therefore, use of a cationic copolymer is a versatile and efficient approach to enhance the operation rate and robustness of toehold-mediated DNA circuits, paving the way for more flexible design and broader application.

2D-3D-Convertible, pH-Responsive Lipid Nanosheets.

2D nanosheets self-assembled with amphiphilic molecules are promising tools for biomedical applications; yet, there are challenges to form and stabilize these nanosheets under complex physiological conditions. Here, the development of lipid nanosheets with high structural stability that can be reversibly converted to cell-sized vesicles by changes in pH within the physiological range robustly, are described. The system is controlled by the membrane disruptive peptide E5 and a cationic copolymer anchored on lipid membranes. It is envisioned that nanosheets formed using the dual anchoring peptide/cationic copolymer system can be employed in dynamic lipidic nanodevices, such as the vesosomes described here, drug delivery systems, and artificial cells.

Autonomous Vesicle/Sheet Transformation of Cell-Sized Lipid Bilayers by Hetero-Grafted Copolymers.

Lipid bilayer transformations are involved in biological phenomena including cell division, autophagy, virus infection, and vesicle transport. Artificial materials to manipulate membrane dynamics play a vital role in cellular engineering and drug delivery technology that accesses the membranes of cells or liposomes. Transformation from 3D lipid vesicles to 2D nanosheets is thermodynamically prohibited because the apolar/polar interfaces between the hydrophobic bilayer edges and water are energetically unfavorable. We recently reported that cell-sized lipid vesicles (or giant vesicles) can be thoroughly transformed to 2D nanosheets by the addition of the amphiphilic E5 peptide and a cationic graft copolymer. Here, to understand the mechanisms underlying the lipid nanosheet formation, we systematically investigated the structural effects of the cationic copolymers on nanosheet formation. We found that lipid nanosheet formation is controlled in an all-or-nothing manner when the graft content of the copolymer is increased from 5.7 mol % to 7.7 mol %. This finding prompted us to obtain autonomous 2D/3D transformation system. A newly designed hetero-grafted cationic copolymers with thermoresponsive poly(N-isopropylacrylamide) grafts enables spontaneous 3D vesicle/2D nanosheet transformation in response to temperature. These findings would enable us to obtain smart nanointerfaces that trigger cell-sized lipid membrane dynamics in response to diverse stimuli and to create 2D-3D convertible lipid-based biomaterials.

Smart Protein Refolding System Based on UCST-Type Ureido Polymers.

We have reported that ureido polymers exhibit upper critical solution temperature (UCST)-type phase behavior in solution, which is the opposite of lower critical solution temperature (LCST)-type behavior. Furthermore, UCST-type ureido polymers undergo liquid-liquid phase separation (LLPS) upon cooling rather than the liquid-solid phase transition of the typical LCST-type polymers. In this study, ureido polymers with hydrophobic groups were prepared to evaluate the effects of cooling-induced LLPS of UCST-type polymers on refolding of proteins. When protein was heated with a ureido polymer functionalized with undecyl groups, aggregation of the protein was prevented. Subsequent cooling incubation resulted in the spontaneous release of the protein from the polymer. The released protein had enzymatic activity, suggesting that the protein refolded properly. Interestingly, efficient refolding was observed when the solution of the UCST-type ureido polymer and protein was incubated at around the phase separation temperature of the polymer, implying that cooling-induced LLPS of the polymer enhanced the release of the protein. Additionally, by centrifugation at 4 °C, the refolded protein was readily separated from the ureido polymers, which precipitated upon cooling.

Cationic Copolymer-Augmented DNA Hybridization Chain Reaction.

Various DNA assembly techniques and structures have emerged with the continuous progress of DNA nanotechnology. DNA hybridization chain reaction (HCR) is a representative example owing to isothermal and enzyme-free features. However, HCR is time consuming and is inhibited by nucleases present in biological samples. Herein, we demonstrated that a cationic copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), significantly facilitated HCR and increased its initiator sensitivity by 40-fold. PLL-g-Dex promoted the generation of HCR products with high molecular weight by accelerating the initiation and the subsequent growth steps of HCR. Moreover, PLL-g-Dex protected the HCR system from nucleases, permitting HCR in the presence of serum components. Addition of PLL-g-Dex is a universal and efficient strategy that does not require optimization of the reactor setup or DNA sequences, thus laying a solid foundation for the wider application of HCR.

Selective Photo-Crosslinking Detection of Methylated Cytosine in DNA Duplex Aided by a Cationic Comb-Type Copolymer.

In the process of cell development and differentiation, C-5-methylation of cytosine (5-methylcytosine: 5-mC) in genome DNA is an important transcriptional regulator that switches between differentiated and undifferentiated states. Further, abnormal DNA methylations are often present in tumor suppressor genes and are associated with many diseases. Therefore, 5-mC detection technology is an important tool in the most exciting fields of molecular biology and diagnosing diseases such as cancers. In this study, we found a novel photo-crosslinking property of psoralen-conjugated oligonucleotide (Ps-Oligo) to the double-stranded DNA (ds-DNA) containing 5-mC in the presence of a cationic comb-type copolymer, poly(allylamine)-graft-dextran (PAA-g-Dex). Photo-crosslinking efficiency of Ps-Oligo to 5-mC in ds-DNA was markedly enhanced in the presence of PAA-g-Dex, permitting 5-mC-targeted crosslinking. We believe that the combination of PAA-g-Dex and Ps-Oligo will be an effective tool for detecting 5-mC in genomic DNA.

A cationic copolymer as a cocatalyst for a peroxidase-mimicking heme-DNAzyme.

Heme binds to a parallel-stranded G-quadruplex DNA to form a peroxidase-mimicking heme-DNAzyme. An interpolyelectrolyte complex between the heme-DNAzyme and a cationic copolymer possessing protonated amino groups was characterized and the peroxidase activity of the complex was evaluated to elucidate the effect of the polymer on the catalytic activity of the heme-DNAzyme. We found that the catalytic activity of the heme-DNAzyme is enhanced through the formation of the interpolyelectrolyte complex due to the general acid catalysis of protonated amino groups of the polymer, enhancing the formation of the iron(IV)oxo porphyrin π-cation radical intermediate known as Compound I. This finding indicates that the polymer with protonated amino groups can act as a cocatalyst for the heme-DNAzyme in the oxidation catalysis. We also found that the enhancement of the activity of the heme-DNAzyme by the polymer depends on the local heme environment such as the negative charge density in the proximity of the heme and substrate accessibility to the heme. These findings provide novel insights as to molecular design of the heme-DNAzyme for enhancing its catalytic activity.

Evaluation of Cooling-Induced Liquid-Liquid Phase Separation of Ureido Polymers as a Cold-Shock Stress Granules Model.

Many intracellular reactions occur in membrane-less organelles that form due to liquid-liquid phase separation (LLPS). Cold-shock stress granules, which are membrane-less organelles, are formed in response to a significant decrease in temperature and recruit biomolecules for regulation of their activities. The authors have reported that synthetic ureido copolymers exhibit cooling-induced LLPS under physiologically relevant conditions. In this study, influences of the cooling-induced LLPS of ureido polymers on enzymatic activity is investigated to evaluate whether the ureido polymers can mimic cold-shock stress granules. The enzyme ß-galactosidase (ß-Gal) is efficiently entrapped into phase-separated coacervates of ureido polymers upon cooling. The activity of ß-Gal is significantly suppressed by the entrapment. The enzymatic activity is recovered after heating, which dissolves the coacervate. Thus, the LLPS formed by ureido polymers are a suitable model for cold-shock stress granules.

Light-Regulated Liquid-Liquid Phase Separation for Spatiotemporal Protein Recruitment and Cell Aggregation.

We have previously shown that the upper critical solution temperature-type thermoresponsive ureido polymers such as polyallylurea and poly(2-ureidoethylmethacrylate) derivatives show liquid-liquid phase separation (LLPS), also known as simple coacervation, under physiological conditions below their phase-separation temperatures (Tp). The addition of the polymer-rich coacervate droplets that result from LLPS to a monolayer cell culture induced aggregation of cells into multicellular spheroids. In this study, we prepared a ureido copolymer, poly(vinylamine-co-vinylurea), with azobenzene substituents (Azo-PVU) and demonstrated light-guided assembly and disassembly of LLPS coacervates. Azo-PVUs with Tp values ranging from 10 to 52 °C were prepared by changing the azobenzene content. Ultraviolet light caused a decrease in the Tp of Azo-PVU because of trans-to-cis photoisomerization of the azobenzene and irradiation with visible light increased the Tp. Thus, LLPS of Azo-PVU was reversibly controlled. The coacervate droplets deposited on a dish surface were immediately dissolved by targeted UV irradiation (owing to a decrease in the Tp). Spatially controlled recruitment of proteins on the dish surface was achieved when protein solution was added to the light-patterned surface. Furthermore, the light-guided deposition of coacervates resulted in the spatiotemporal transformation of monolayer cells to aggregates. This light-controlled LLPS will allow the preparation of novel liquid-based materials for biomolecular and cellular engineering.

Crystallization-induced mechanofluorescence for visualization of polymer crystallization.

The growth of lamellar crystals has been studied in particular for spherulites in polymeric materials. Even though such spherulitic structures and their growth are of crucial importance for the mechanical and optical properties of the resulting polymeric materials, several issues regarding the residual stress remain unresolved in the wider context of crystal growth. To gain further insight into micro-mechanical forces during the crystallization process of lamellar crystals in polymeric materials, herein, we introduce tetraarylsuccinonitrile (TASN), which generates relatively stable radicals with yellow fluorescence upon homolytic cleavage at the central C-C bond in response to mechanical stress, into crystalline polymers. The obtained crystalline polymers with TASN at the center of the polymer chain allow not only to visualize the stress arising from micro-mechanical forces during polymer crystallization via fluorescence microscopy but also to evaluate the micro-mechanical forces upon growing polymer lamellar crystals by electron paramagnetic resonance, which is able to detect the radicals generated during polymer crystallization.

Spatially regulated activation of membrane fusogenic peptides with chaperone-like ionic copolymers.

Controlled or targeted membrane lysis induced by cascades of assembly and activation of biomolecules on membrane surfaces is important in programmed cell death and host defense systems. In a previous study, we reported that an ionic graft copolymer with a polycation backbone and water-soluble graft chains, poly(allylamine)-graft-dextran (PAA-g-Dex) chaperoned folding and assembly of E5, a membrane-destructive peptide derived from influenza hemagglutinin, to its increase membrane-disruptive activity. In this study, we modified the copolymer with long acyl chains, which resulted in delivery of the copolymer to membrane surfaces of liposomes and living cells. The liposomes with PAA-g-Dex functionalized with stearic acid (PAA-g-Dex-SA) on their surfaces underwent vesicle-to-sheet conversion upon addition of E5, whereas control liposomes did not. E5 also induced selective lysis of cells incubated with PAA-g-Dex-SA. The spatially specific activation of E5 on target membrane surfaces driven by self-assembly of copolymer and activation of E5 should find application in lipid-based delivery devices and cell-based therapeutics.

One-step isothermal RNA detection with LNA-modified MNAzymes chaperoned by cationic copolymer.

RNA detection permits early diagnosis of several infectious diseases and cancers, which prevent propagation of diseases and improve treatment efficacy. However, standard technique for RNA detection such as reverse transcription-quantitative polymerase chain reaction has complicated procedure and requires well-trained personnel and specialized lab equipment. These shortcomings limit the application for point-of-care analysis which is critical for rapid and effective disease management. The multicomponent nucleic acid enzymes (MNAzymes) are one of the promising biosensors for simple, isothermal and enzyme-free RNA detection. Herein, we demonstrate simple yet effective strategies that significantly enhance analytical performance of MNAzymes. The addition of the cationic copolymer and structural modification of MNAzyme significantly enhanced selectivity and activity of MNAzymes by 250 fold and 2,700 fold, respectively. The highly simplified RNA detection system achieved a detection limit of 73 fM target concentration without additional amplification. The robustness of MNAzyme in the presence of non-target RNA was also improved. Our finding opens up a route toward the development of an alternative rapid, sensitive, isothermal, and protein-free RNA diagnostic tool, which expected to be of great clinical significance.

Cationic copolymer enhances 8-17 DNAzyme and MNAzyme activities.

DNAzymes are DNA molecules capable of catalytic activity. The catalytic core of DNAzymes can be separated and conjugated with target binding arms to create allosteric DNAzymes known as multi-component nucleic acid enzymes (MNAzymes). Two widely used DNAzymes are the 10-23 and the 8-17 DNAzymes. These DNAzymes differ in catalytic core structures, cleavage sites, and reactive metal ion cofactors. Previously we showed that the presence of a cationic comb-type polymer poly(l-lysine)-graft-dextran (PLL-g-Dex) improved activities of the 10-23 DNAzyme and MNAzyme by facilitating assembly of the catalytic complex. In this work, we demonstrate that PLL-g-Dex enhances activities of the 8-17 DNAzyme and MNAzyme; poly(allylamine)-graft-dextran and cationic homopolymers did not enhance activities. Metal ion and pH dependences were observed in the presence of PLL-g-Dex, suggesting that the cationic copolymer did not impede the interaction between the metal ion and the DNA-based enzymes. Thus, PLL-g-Dex has chaperone-like activity for DNAzymes and MNAzymes regardless of structures, cleavage sites, and cofactors.

Bundling Process of Citrulline Polypeptides upon UCST-Type Phase Separation.

Ureido-modified poly(l-citrulline) (l-ornithine-co-l-citrulline denoted by PlOC) shows UCST-type phase separation behavior even under physiologically relevant conditions, which forms an α-helix structure above its phase separation temperature (Tp) but transforms into a solid-like aggregation composed of regular hexagonal packed cylinders below the Tp. This morphological transformation is characteristic of the phase separation behavior, but the mechanism behind it has remained incompletely understood. Here, we studied the phase separation behavior using small-angle X-ray scattering (SAXS) measurements. To analyze the SAXS data, we employed the modified unified model proposed previously, which decomposes the scattering profile into each structural element, such as the α-helices and their aggregation formed via hydrogen-bonding interactions between the ureido groups. The aggregation level is dependent on the temperature (T) and grouped into three classes: (1) mass-fractal aggregation composed of the α-helix (T > Tp), (2) spherical aggregation composed of the hexagonal packed cylinder (T < Tp), and (3) micro-order agglomeration formed by mutual fusion of the spherical aggregation, which appears as a solid-like aggregation. The SAXS analysis suggested that the transformation from the dispersed state as the α-helix to the agglomeration containing hierarchical structures occurs in a stepwise manner when the temperature falls below the Tp, which might also be transition behavior similar to the process of protein folding through folding intermediates.

Cationic copolymer-chaperoned short-armed 10-23 DNAzymes.

The cationic copolymer poly(L-lysine)-graft-dextran (PLL-g-Dex) has nucleic acid chaperone-like activity. The copolymer facilitates both DNA hybridization and strand exchange reactions. For these reasons, DNA-based enzyme (DNAzyme) activity is enhanced in the presence of copolymer. In this study, we evaluated activities of DNAzymes with substrate-binding arms (S-arms) of various lengths. The copolymer promoted DNAzyme reactivity and turnover efficacy, and, depending on S-arm length, maximally accelerated the reaction rate by 250-fold compared to the rate in the absence of copolymer. The copolymer permitted up to six nucleotides truncation of the S-arms having initial length of 10 and 11 nucleotides without loss of catalytic efficiency, enable tuning of the optimal temperature ranging from 30 to 55 °C. The approach might be useful for the development of DNAzyme systems targeting short or highly structured RNAs as well for improvement of DNAzyme-based nanomachines and biosensors.

Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection.

Multi-component nucleic acid enzymes (MNAzymes) are allosteric deoxyribozymes that are activated upon binding of a specific nucleic acid effector. MNAzyme activity is limited due to an insufficient assembly of the MNAzyme and its turnover. In this work, we describe the successful improvement of MNAzyme reactivity and selectivity by addition of cationic copolymers, which exhibit nucleic acid chaperone-like activity. The copolymer allowed a 210-fold increase in signal activity and a 95-fold increase in the signal-to-background selectivity of MNAzymes constructed for microRNA (miRNA) detection. The selectivity of the MNAzyme for homologous miRNAs was demonstrated in a multiplex format in which isothermal reactions of two different MNAzymes were performed. In addition, the copolymer permitted miRNA detections even in the presence of a ribonuclease which is ubiquitous in environments, indicating the protective effect of the copolymer against ribonucleases.

Cationic Copolymer-Chaperoned 2D-3D Reversible Conversion of Lipid Membranes.

Nanosheets have thicknesses on the order of nanometers and planar dimensions in the micrometer range. Nanomaterials that are capable of converting reversibly between 2D nanosheets and 3D structures in response to specific triggers can enable construction of nanodevices. Supra-molecular lipid nanosheets and their triggered conversions to 3D structures including vesicles and cups are reported. They are produced from lipid vesicles upon addition of amphiphilic peptides and cationic copolymers that act as peptide chaperones. By regulation of the chaperoning activity of the copolymer, 2D to 3D conversions are reversibly triggered, allowing tuning of lipid bilayer structures and functionalities.