Hydrogel‐Immobilized Supercharged Proteins

Campbell, E.C.; Grant, Jacob; Wang, Yi; Sandhu, Mahakaran; Williams, Richard J.; Nisbet, David R.; Perriman, Adam W.; Lupton, David W.; Jackson, Colin J.

doi:10.1002/adbi.201700240

Cited by 17 publications

(15 citation statements)

References 71 publications

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“…56 Several groups have also recently reported on the effects of microenvironment on enzyme activity and specifically that of adding charge near the enzyme active site, which would presumably also affect local ionic character and pH. 57,58 This may well mimic the effect of DNA in the current scenario. Lastly, it is well known that enzyme activity can often be enhanced by attachment to nanoobjects, e.g., nanoparticles, with various mechanisms implicated including stabilization of multimeric enzymes and facilitation of product dissociation.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

et al. 2019

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Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway’s kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

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Section: Resultsmentioning

confidence: 99%

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

et al. 2019

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“…For example, a series of recent studies developed and optimized a PTE‐DNA‐Au nanoparticle system that enhances k cat by altering the reaction mechanism (Breger et al, 2015; Breger et al, 2017; Samanta et al, 2018). PTE‐Protein hydrogel systems also modified reaction kinetics by altering the local chemical environment (Campbell et al, 2018; Lu, Wheeldon, & Banta, 2010). Our approach uses molecular scaffolds to add new chemical and physical features to the surface of an enzyme.…”

Section: Resultsmentioning

confidence: 99%

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Lang

Hong

Baker

et al. 2020

Biotech & Bioengineering

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Kinetic enhancement of organophosphate hydrolysis is a long‐standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl‐parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in KM and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased KM by 11‐fold, with a corresponding increase in second‐order rate constant. The initial rates of VX and methyl‐parathion hydrolysis were also increased by 3.1‐ and 6.7‐fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE‐DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.

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“…Previously, flow-induced hydrodynamic shear stress has also increased EMT in 2D and 3D lung-cancer models, thus, future investigations may wish to incorporate a more effective model of measuring the 3D migration of the cells, such as a modified transwell system, its development into a bioprinted system [ 60 ] or a microfluidic platform to further access cell mobility and drug effectiveness within an artificial TME [ 61 ] The precise nature of the migrating cells and the EMT such as features of the system can be further explored via the modular approach we have detailed here. For example, other proteins [ 62 , 63 ] and biopolymers [ 64 ] can be coassembled to the system, or a population of healthy cells can be cultured in 3D before the addition of a spheroid. The lung tumour model detailed within this study could be used to further explore effective treatment of lung cancer via more tumour-like conditions, drug delivery and a model of cell mobility.…”

Section: Discussionmentioning

confidence: 99%

Self-Assembled Peptide Habitats to Model Tumor Metastasis

Balushi

Boyd‐Moss

Samarasinghe

et al. 2022

Gels

Self Cite

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Metastatic tumours are complex ecosystems; a community of multiple cell types, including cancerous cells, fibroblasts, and immune cells that exist within a supportive and specific microenvironment. The interplay of these cells, together with tissue specific chemical, structural and temporal signals within a three-dimensional (3D) habitat, direct tumour cell behavior, a subtlety that can be easily lost in 2D tissue culture. Here, we investigate a significantly improved tool, consisting of a novel matrix of functionally programmed peptide sequences, self-assembled into a scaffold to enable the growth and the migration of multicellular lung tumour spheroids, as proof-of-concept. This 3D functional model aims to mimic the biological, chemical, and contextual cues of an in vivo tumor more closely than a typically used, unstructured hydrogel, allowing spatial and temporal activity modelling. This approach shows promise as a cancer model, enhancing current understandings of how tumours progress and spread over time within their microenvironment.

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Hydrogel‐Immobilized Supercharged Proteins

Abstract: The remarkable catalytic potential of enzymes in chemical synthesis, environmental bioremediation and medical therapeutics is limited by their longevity and stability.

Cited by 17 publications

References 71 publications

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

Enhanced Catalysis from Multienzyme Cascades Assembled on a DNA Origami Triangle

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Self-Assembled Peptide Habitats to Model Tumor Metastasis

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