Molecular Sequestration Research Articles

Engineering Sequestration-Based Biomolecular Classifiers with Shared Resources.

Constructing molecular classifiers that enable cells to recognize linear and nonlinear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effects of biological constraints such as resource limitation and competitive binding on the function of those classifiers have been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working based on the principles of molecular sequestration between the sigma factor and its antisigma molecule. We then investigate how the output of the biomolecular perceptron, i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multilayer perceptron neural networks and outline design principles that enable the construction of nonlinear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects.

Chemical reaction motifs driving non-equilibrium behaviours in phase separating materials.

Chemical reactions that couple to systems that phase separate have been implicated in diverse contexts from biology to materials science. However, how a particular set of chemical reactions (chemical reaction network, CRN) would affect the behaviours of a phase separating system is difficult to fully predict theoretically. In this paper, we analyse a mean field theory coupling CRNs to a combined system of phase separating and non-phase separating materials and analyse how the properties of the CRNs affect different classes of non-equilibrium behaviour: microphase separation or temporally oscillating patterns. We examine the problem of achieving microphase separated condensates by statistical analysis of the Jacobians, of which the most important motifs are negative feedback of the phase separating component and combined inhibition/activation by the non-phase separating components. We then identify CRN motifs that are likely to yield microphase by examining randomly generated networks and parameters. Molecular sequestration of the phase separating motif is shown to be the most robust towards yielding microphase separation. Subsequently, we find that dynamics of the phase separating species is promoted most easily by inducing oscillations in the diffusive components coupled to the phase separating species. Our results provide guidance towards the design of CRNs that manage the formation, dissolution and organization of compartments.

Photoinduced phase transition between vesicles and coacervates using low-molecular-weight catanionic amphiphiles

Amphiphiles form various molecular self-assemblies in the aqueous phase, such as vesicles and coacervates, depending on molecular properties. Recently, phase transitions between vesicles and coacervates have drawn much attention because the switching between “closed” and “open” compartments could achieve the controllability of molecular sequestrations. We expect that the photoinduced drastic change in the molecular structure of catanionic amphiphiles comprising azobenzene-containing cationic amphiphiles (Azo) and anionic sodium dodecyl sulfate causes the transition. Self-assemblies prepared by a thin-film hydration method were observed using microscopy under UV and subsequent visible-light illumination. Additionally, investigation of selectivity for molecular sequestration and assay of enzymatic reactions in the self-assemblies were conducted. Photoinduced reversible phase transitions between vesicles and coacervates occurred owing to the photoisomerization of Azo. The formed coacervates had selectivity for molecular sequestration. Additionally, the transition achieved fusion of different vesicles via coacervates. Although coacervates are generally known to facilitate enzymatic reactions, the reactions were inhibited in the presence of the formed self-assemblies due to the deactivation of enzymes. Our results represent rapid and non-invasive systems of reversible vesicle–coacervate transitions. Furthermore, the findings provide a new aspect of coacervates in which catanionic low-molecular-weight amphiphiles could act as suppressors of chemical reactions.

Spatial control of self-organizing vascular networks with programmable aptamer-tethered growth factor photopatterning.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800​μm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

The Pol III transcriptome: Basic features, recurrent patterns, and emerging roles in cancer.

The RNA polymerase III (Pol III) transcriptome is universally comprised of short, highly structured noncoding RNA (ncRNA). Through RNA-protein interactions, the Pol III transcriptome actuates functional activities ranging from nuclear gene regulation (7SK), splicing (U6, U6atac), and RNA maturation and stability (RMRP, RPPH1, Y RNA), to cytoplasmic protein targeting (7SL) and translation (tRNA, 5S rRNA). In higher eukaryotes, the Pol III transcriptome has expanded to include additional, recently evolved ncRNA species that effectively broaden the footprint of Pol III transcription to additional cellular activities. Newly evolved ncRNAs function as riboregulators of autophagy (vault), immune signaling cascades (nc886), and translation (Alu, BC200, snaR). Notably, upregulation of Pol III transcription is frequently observed in cancer, and multiple ncRNA species are linked to both cancer progression and poor survival outcomes among cancer patients. In this review, we outline the basic features and functions of the Pol III transcriptome, and the evidence for dysregulation and dysfunction for each ncRNA in cancer. When taken together, recurrent patterns emerge, ranging from shared functional motifs that include molecular scaffolding and protein sequestration, overlapping protein interactions, and immunostimulatory activities, to the biogenesis of analogous small RNA fragments and noncanonical miRNAs, augmenting the function of the Pol III transcriptome and further broadening its role in cancer. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Processing of Small RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Building Subtraction Operators and Controllers via Molecular Sequestration

Building Subtraction Operators and Controllers via Molecular Sequestration

Dichotomous feedback: a signal sequestration-based feedback mechanism for biocontroller design

We introduce a new design framework for implementing negative feedback regulation in synthetic biology, which we term ‘dichotomous feedback’. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the process’s architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realize than ‘molecular sequestration’ and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where a second response regulator could be competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

Development of a Biomimetic Extracellular Matrix with Functions of Protein Sequestration and Cell Attachment Using Dual Aptamer-Functionalized Hydrogels.

The extracellular matrix (ECM) has not only cell-binding sites for cell attachment but also protein-binding sites for molecular sequestration. Aptamers have high binding affinities and specificities against their target molecules. Thus, the purpose of this work was to develop dual aptamer-functionalized hydrogels for simultaneously recapitulating the two key features of the ECM in binding cells and sequestering proteins. We synthesized the hydrogels using free-radical polymerization in a freezing procedure. As the hydrogels were macroporous with pores of 40-50 μm, both cells and proteins could be loaded into the hydrogels after the synthesis. Importantly, the vascular endothelial growth factor (VEGF) aptamer improved VEGF sequestration and reduced the apparent diffusivity of VEGF by over 2 orders of magnitude, resultantly prolonging VEGF retention and release. The c-MET aptamer promoted the attachment of endothelial cells in the hydrogel network. When two aptamers were both incorporated into the hydrogel, they could produce synergistic effects on cell survival and growth. Thus, this work has successfully demonstrated the potential of developing biomimetic ECMs with two key functions of cell attachment and protein sequestration using dual aptamer-functionalized hydrogels.

Ultrasensitive molecular controllers for quasi-integral feedback.

Feedback control has enabled the success of automated technologies by mitigating the effects of variability, unknown disturbances, and noise. While it is known that biological feedback loops reduce the impact of noise and help shape kinetic responses, many questions remain about how to design molecular integral controllers. Here, we propose a modular strategy to build molecular quasi-integral feedback controllers, which involves following two design principles. The first principle is to utilize an ultrasensitive response, which determines the gain of the controller and influences the steady-state error. The second is to use a tunable threshold of the ultrasensitive response, which determines the equilibrium point of the system. We describe a reaction network, named brink controller, that satisfies these conditions by combining molecular sequestration and an activation/deactivation cycle. With computational models, we examine potential biological implementations of brink controllers, and we illustrate different example applications.

Evaluation of potassium glycinate, potassium lysinate, potassium sarcosinate and potassium threonate solutions in CO2 capture using membranes

The mitigation process of greenhouse gases emission such as CO2 into the atmosphere is known as a vital necessity in modern societies. Nowadays, amino acid salt solutions (AASSs) have been extensively applied as a promising alternative to alkanol amine absorbents to increase the CO2 sequestration efficiency from disparate gaseous flows. This article aims to computationally and theoretically evaluate the CO2 separation percentage using potassium glycinate (PG), potassium lysinate (PL), potassium sarcosinate (PS) and potassium threonate (PT) amino acid solutions from an inlet gaseous mixture inside a hydrophobic membrane contactor (HMC). To do this, the governing first principal equations inside the HMC are solved using the computational fluid dynamics procedure based on finite element technique. Acceptable agreement between the simulation results and experimental values with average deviation of approximately 3% implies the validation of developed two-dimensional (2D) simulation approach developed in this study. The analysis of obtained results demonstrated that PG is the most efficient amino acid solution for CO2 molecular sequestration with the ability of separating 90% of inlet CO2 in the system. The order of solutions is 90% sequestration using PG > 89.3% sequestration using PT > 77.4% sequestration using PL > 72.3% sequestration using PS.

Computational fluid dynamics simulation of NO2 molecular sequestration from a gaseous stream using NaOH liquid absorbent through porous membrane contactors

Nitrogen dioxide (NO2) is known as a detrimental acidic contaminant, which its emission into the atmosphere can significantly endanger the human well-being. Therefore, the necessity of mitigating and controlling the NO2 molecules emission needs substantial attention. This research aims to addresses molecular sequestration of NO2 from a gaseous flow applying NaOH absorbent in a hydrophobic porous membrane contactor (HPMC). A mechanistic two dimensional (2D) model is developed based on mass transfer and fluid flow, while computational fluid dynamics (CFD) approach is used for numerical solution of the model's equations. The governing mathematical equations with their corresponding boundary conditions are solved via finite element (FE) approach. The comparison of the model's findings with the experimental data corroborated an excellent agreement with average absolute relative error (AARE) of approximately 2%. The results also demonstrated that the NaOH liquid absorbent can sequester 90% of the NO2 molecules at the inlet feed gas stream, which implies NaOH adequacy for NO2 molecular sequestration. Additionally, the results demonstrated the improvement of NO2 molecular sequestration by increasing some functional/operational parameters such as inner tube radius, module length, absorbent concentration and gas flow velocity inside the HPMC.

Thermodynamic Evidence of Structural Transformations in CO2-Loaded Metal-Organic Framework Zn(MeIm)2 from Heat Capacity Measurements.

Metal-organic frameworks are a class of porous compounds with potential applications in molecular sieving, gas sequestration, and catalysis. One family of MOFs, zeolitic imidizolate frameworks (ZIFs), is of particular interest for carbon dioxide sequestration. We have previously reported the heat capacity of the sodalite topology of the zinc 2-methylimidazolate framework (ZIF-8), and in this Article we present the first low-temperature heat capacity measurements of ZIF-8 with various amounts of sorbed CO2. Molar heat capacities from 1.8 to 300 K are presented for samples containing up to 0.99 mol of CO2 per mol of ZIF-8. Samples with at least 0.56 mol of CO2 per mol of ZIF-8 display a large, broad anomaly from 70 to 220 K with a shoulder on the low-temperature side, suggesting sorption-induced structural transitions. We attribute the broad anomaly partially to a gate-opening transition, with the remainder resulting from CO2 rearrangement and/or lattice expansion. The measurements also reveal a subtle anomaly from 0 to 70 K in all samples that does not exist in the sorbate-free material, which likely reflects new vibrational modes resulting from sorbate/ZIF-8 interactions. These results provide the first thermodynamic evidence of structural transitions induced by CO2 sorption in the ZIF-8 framework.

Removal of the synthetic hormone methyltestosterone from aqueous solution using a β-cyclodextrin/silica composite

Contamination of water with steroid residues can cause a number of environmental damages, affecting exposed organisms including man. The development of technologies for treatment or removal of this type of micropollutant from water is of paramount importance. In this study, citric acid was used to functionalize β-Cyclodextrin (bCD) on the silica surface generating an organic-inorganic hybrid composite for application in molecular sequestration. The functionalization percentage was high, with about 62.6% of the composite mass corresponding to the organic part of the material. 13C NMR and infrared spectroscopic analysis indicate that the functionalization mechanism occurs by an esterification reaction between the citric acid with the silanol groups from silica and the primary hydroxyls of the bCDs. Fast adsorption of the methyltestosterone steroid was observed at acid pH, with a high adsorption capacity of 11 mg g−1. The best kinetic and isotherm models fit indicated that the adsorption occurred by a physical mechanism at independent sites with the steroid molecule possibly captured by two bCDs. The removal process was spontaneous and exothermic, with the existence of weak interactions between the hormone and the composite, and its regeneration is quite fast efficient with the displacement of the complexation equilibrium. The results obtained in this study demonstrate the considerable potential of the composite for use in the treatment of wastewater containing the steroid studied, and its efficacy should be evaluated for other steroid molecules.

Heat capacity and thermodynamic functions of crystalline forms of the metal-organic framework zinc 2-methylimidazolate, Zn(MeIm)2

Zeolitic imidazolate frameworks (ZIFs) are composed of metal atoms connected with imidazole-like linkers, and these frameworks have potential for applications in molecular sieving, gas sequestration, and catalysis. In addition, these materials form true polymorphs with the same chemical composition but different topologies. In this paper, we present the results of low temperature heat capacity and inelastic neutron scattering studies of the sodalite (SOD) and diamondoid (dia) topologies of the popular zinc 2-methylimidazolate framework, Zn(MeIm)2. Molar heat capacities from 1.8 K to 300 K are presented, along with theoretical fits and the values of Cp,m°, Δ0TSm°, Δ0THm°, andΦm°calculated from those fits. The Gibbs energy of the transformation from SOD to dia is −(4.6 ± 2.2) kJ, and this transformation is primarily enthalpically driven. The results of this study are compared with previous measurements on the zinc 2-ethylimidazolate framework, Zn(EtIm)2. Inelastic neutron scattering measurements confirm the presence of low energy modes and suggest that the higher heat capacity of SOD at low temperatures is due to the dynamics of the methyl groups on the methylimidazolate linkers.

A Singular Singular Perturbation Problem Arising From a Class of Biomolecular Feedback Controllers

A long-standing challenge in synthetic biology is to engineer biomolecular systems that can perform robustly in highly uncertain cellular environments. Recently, there has been increasing interest to design biomolecular feedback controllers to address this challenge. Molecular sequestration is one of the proposed feedback mechanisms. For this type of design, when all reactions within the controller are sufficiently fast, the process output can reach a set-point regardless of parametric uncertainties. However, as we demonstrate in this letter, the way in which molecular sequestration affects the fast controller dynamics leads to a singular singularly perturbed (SSP) system. In an SSP system, the boundary layer Jacobian is singular and thus standard singular perturbation approaches cannot be applied, posing difficulties to analytically determine the performance of sequestration-based controllers. In this letter, we consider a class of linear systems that capture the key structure of sequestration-based controllers. We show that, under certain technical conditions, these SSP systems can still be approximated by reduced-order systems that are dependent on the small parameter. This result allows us to analytically evaluate the tracking performance of the linearized model of a sequestration-based controller.

Molecular sequestration mechanisms of heavy metals by iron oxides in soils using synchrotronbased techniques: A review

Iron oxides, widely distributed in soils, have large specific surface areas and strong affinity to heavy metals, and thus play a significant role in the transformation of heavy metals in soils. To understand the environmental behaviors of heavy metals and assess their bioavailability in contaminated soils, it is important to investigate the sequestration mechanisms of heavy metals by iron oxides. The traditional methods, including adsorption modeling and chemical extraction fractionation, have obvious limitations and provide little information on the sequestration mechanisms of heavy metal by iron oxides at the molecular level. The application of synchrotron-based techniques in environmental soil science has greatly enhanced the molecular-level understanding of the immobilization mechanisms of heavy metals by iron oxides under various environmental conditions. Here, we reviewed the development of synchrotron-based techniques and summarized the molecular sequestration mechanisms of heavy metals by iron oxides in model and real soil systems under various environmental factors. The future development of synchrotron-based techniques and their applications were prospected.

Stochastic sequestration dynamics: a minimal model with extrinsic noise for bimodal distributions and competitors correlation

Many biological processes are known to be based on molecular sequestration. This kind of dynamics involves two types of molecular species, namely targets and sequestrants, that bind to form a complex. In the simple framework of mass-action law, key features of these systems appear to be threshold-like profiles of the amounts of free molecules as a function of the parameters determining their possible maximum abundance. However, biochemical processes are probabilistic and take place in stochastically fluctuating environments. How these different sources of noise affect the final outcome of the network is not completely characterised yet. In this paper we specifically investigate the effects induced by a source of extrinsic noise onto a minimal stochastic model of molecular sequestration. We analytically show how bimodal distributions of the targets can appear and characterise them as a result of noise filtering mediated by the threshold response. We then address the correlations between target species induced by the sequestrant and discuss how extrinsic noise can turn the negative correlation caused by competition into a positive one. Finally, we consider the more complex scenario of competitive inhibition for enzymatic kinetics and discuss the relevance of our findings with respect to applications.

Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression.

Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.

Programmable hydrogels

Programmable hydrogels are defined as hydrogels that are able to change their properties and functions periodically, reversibly and/or sequentially on demand. They are different from those responsive hydrogels whose changes are passive or cannot be stopped or reversed once started and vice versa. The purpose of this review is to summarize major progress in developing programmable hydrogels from the viewpoints of principles, functions and biomedical applications. The principles are first introduced in three categories including biological, chemical and physical stimulation. With the stimulation, programmable hydrogels can undergo functional changes in dimension, mechanical support, cell attachment and molecular sequestration, which are introduced in the middle of this review. The last section is focused on the introduction and discussion of four biomedical applications including mechanistic studies in mechanobiology, tissue engineering, cell separation and protein delivery.

Principles and Applications of Functional Lipidic Biomaterials in Molecular Recognition, Membrane Protein Crystallization and Drug Delivery

We have designed and synthesized a library of lipids with novel functionalities in order to correlate lipid molecular structure with the ensuing nanomaterial dynamics, stability and phase behavior. The designed lipids were assembled as within lipidic mesophases and their dispersions, forming functional biomaterials that were employed in areas ranging from molecular recognition, sequestration and enrichment of nucleic acids to biosensing. An exciting new class of biomaterials that are based on cyclopropanated lipids was developed. These form stable lipidic cubic phases (LCPs) at low temperature, opening the way to conduct biophysical and biochemical investigations on temperature sensitive bio-macromolecules, specifically membrane proteins. Furthermore, pH-sensitive lipidic matrices for hydrophilic as well as hydrophobic drug incorporation and release were designed, as well as stimuli-responsive lipids that can be incorporated into biomaterials. Efficient pH- and light-induced binding, release and sequestration of hydrophilic dyes were demonstrated. Significantly, these processes could be activated sequentially, thereby achieving high degree of temporal and dosage control. The scope of lipidic materials for drug delivery was expanded to azobenzene-containing hexagonal phases, in which “on demand” single-step as well as sequential light-triggered release and retention of embedded dye molecules were demonstrated. Finally, cubosomes were stabilized and functionalized with a novel, designed biotin-based block copolymer, resulting in dispersed biomaterials that were applied against the human adenocarcinoma cell line HeLa. These cubosomes are able to simultaneously transport paclitaxel, a potent anti-cancer drug, and a hydrophobic fluorescent dye in active targeting of cancer cells. Such biotinylated cubosomes are potentially applicable in diagnosis, drug delivery and monitoring of therapeutic response for active targeting versus cancer cells.