scholarly journals Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets

2020 ◽  
Vol 6 (23) ◽  
pp. eaba3471 ◽  
Author(s):  
Yusuke Sato ◽  
Tetsuro Sakamoto ◽  
Masahiro Takinoue
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Macromolecular Structure ◽  
Base Sequence ◽  
Enzymatic Reactions ◽  
Dna Nanostructures ◽  
Sequence Design ◽  
Molecular Systems ◽  
Liquid Liquid Phase Separation

DNA has the potential to achieve a controllable macromolecular structure, such as hydrogels or droplets formed through liquid-liquid phase separation (LLPS), as the design of its base sequence can result in programmable interactions. Here, we constructed “DNA droplets” via LLPS of sequence-designed DNA nanostructures and controlled their dynamic functions by designing their sequences. Specifically, we were able to adjust the temperature required for the formation of DNA droplets by designing the sequences. In addition, the fusion, fission, and formation of Janus-shaped droplets were controlled by sequence design and enzymatic reactions. Furthermore, modifications of proteins with sequence-designed DNAs allowed for their capture into specific droplets. Overall, our results provide a platform for designing and controlling macromolecular droplets via the information encoded in component molecules and pave the way for various applications of sequence-designed DNA such as cell mimics, synthetic membraneless organelles, and artificial molecular systems.

scholarly journals Dynamic Formation of Liquid Droplets Triggered by Sequential Enzymatic Reactions

2020 ◽  
Author(s):  
Tomoto Ura ◽  
Shunsuke Tomita ◽  
Kentaro Shiraki
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Enzyme Reaction ◽  
Model System ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Dynamic Formation ◽  
Liquid Liquid Phase Separation

<p>A model system was developed that dynamically generates two different liquid droplets via liquid–liquid phase separation coupled with a sequential glycolytic reaction. The sequential two-enzyme reaction triggers the formation/dissolution of the liquid droplets. The droplets, in turn, compartmentalize each enzymatic step and generate feedback to accelerate the overall reaction.</p>

scholarly journals Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

2020 ◽  
Author(s):  
Tomoto Ura ◽  
Ako Kagawa ◽  
Hiromasa Yagi ◽  
Naoya Tochio ◽  
Takanori Kigawa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Living Cells ◽  
Enzyme Activation ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Essential Information ◽  
Liquid Liquid Phase Separation

ABSTRACTLiquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

scholarly journals Regulating enzymatic reactions in Escherichia coli utilizing light-responsive cellular compartments based on liquid-liquid phase separation

2020 ◽  
Author(s):  
Zikang Huang ◽  
Lize Sun ◽  
Genzhe Lu ◽  
Hongrui Liu ◽  
Zihan Zhai ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Small Organic Molecules ◽  
E Coli ◽  
System A ◽  
Control Target ◽  
Cellular Compartments ◽  
Liquid Liquid Phase Separation

AbstractEnzymatic reactions in cells are well organized into different compartments, among which protein-based membraneless compartments formed through liquid-liquid phase separation (LLPS) are believed to play important roles1,2. Hijacking them for our own purpose has promising applications in metabolic engineering3. Yet, it is still hard to precisely and dynamically control target enzymatic reactions in those compartments4. To address those problems, we developed Photo-Activated Switch in E. coli (PhASE), based on phase separating scaffold proteins and optogenetic tools. In this system, a protein of interest (POI) can be enriched up to 15-fold by LLPS-based compartments from cytosol within only a few seconds once activated by light, and become fully dispersed again within 15 minutes. Furthermore, we explored the potentiality of the LLPS-based compartment in enriching small organic molecules directly via chemical-scaffold interaction. With enzymes and substrates co-localized under light induction, the overall reaction efficiency could be enhanced. Using luciferin and catechol oxidation as model enzymatic reactions, we found that they could accelerate 2.3-fold and 1.6-fold, respectively, when regulated by PhASE. We anticipate our system to be an extension of the synthetic biology toolkit, facilitating rapid recruitment and release of POIs, and reversible regulation of enzymatic reactions.

scholarly journals Pattern Regulation of DNA Hydrogels Formed by Lateral Phase Separation of DNA Nanostructures on Water-in-Oil Droplet Interfaces

2021 ◽  
Author(s):  
Yusuke Sato ◽  
Masahiro Takinoue
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Solution Temperature ◽  
Dna Nanostructures ◽  
Artificial Cell ◽  
Lateral Phase Separation ◽  
Hydrogel Capsules ◽  
Liquid Liquid Phase Separation ◽  
Dna Hydrogels

<p>Phase separation is a key phenomenon in artificial cell construction. Recent studies have shown that the liquid-liquid phase separation of designed-DNA nanostructures induces the formation of liquid-like condensates that eventually become hydrogels by lowering the solution temperature. As a compartmental capsule is an essential artificial cell structure, many studies have focused on the lateral phase separation of artificial lipid vesicles. However, controlling phase separation using a molecular design approach remains challenging. Here, we present the lateral liquid-liquid phase separation of DNA nanostructures that leads to the formation of phase-separated capsule-like hydrogels. We designed three types of DNA nanostructures (two orthogonal and a linker nanostructure) that were adsorbed onto an interface of water-in-oil droplets via electrostatic interactions. The phase separation of DNA nanostructures led to the formation of hydrogels of bicontinuous, patch, and mix patterns, due to the immiscibility of liquid-like DNA during the self-assembly process. The frequency of appearance of these patterns was regulated by designing DNA sequences and altering the mixing ratio of the nanostructures. We constructed a phase diagram for the capsule-like DNA hydrogels by investigating pattern formation under various conditions. Our results provide a method for the design and control of phase-separated hydrogel capsules using sequence-designed DNAs. We envision that by incorporating various DNA nanodevices into DNA hydrogel capsules, the capsules will gain molecular sensing, chemical-information processing, and mechano-chemical actuating functions, allowing the construction of functional molecular systems.</p>

scholarly journals Dynamic Formation of Liquid Droplets Triggered by Sequential Enzymatic Reactions

2020 ◽  
Author(s):  
Tomoto Ura ◽  
Shunsuke Tomita ◽  
Kentaro Shiraki
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Enzyme Reaction ◽  
Model System ◽  
Liquid Phase Separation ◽  
Enzymatic Reactions ◽  
Liquid Droplets ◽  
Dynamic Formation ◽  
Liquid Liquid Phase Separation

<p>A model system was developed that dynamically generates two different liquid droplets via liquid–liquid phase separation coupled with a sequential glycolytic reaction. The sequential two-enzyme reaction triggers the formation/dissolution of the liquid droplets. The droplets, in turn, compartmentalize each enzymatic step and generate feedback to accelerate the overall reaction.</p>

scholarly journals Pattern Regulation of DNA Hydrogels Formed by Lateral Phase Separation of DNA Nanostructures on Water-in-Oil Droplet Interfaces

2021 ◽  
Author(s):  
Yusuke Sato ◽  
Masahiro Takinoue
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Solution Temperature ◽  
Dna Nanostructures ◽  
Artificial Cell ◽  
Lateral Phase Separation ◽  
Hydrogel Capsules ◽  
Liquid Liquid Phase Separation ◽  
Dna Hydrogels

<p>Phase separation is a key phenomenon in artificial cell construction. Recent studies have shown that the liquid-liquid phase separation of designed-DNA nanostructures induces the formation of liquid-like condensates that eventually become hydrogels by lowering the solution temperature. As a compartmental capsule is an essential artificial cell structure, many studies have focused on the lateral phase separation of artificial lipid vesicles. However, controlling phase separation using a molecular design approach remains challenging. Here, we present the lateral liquid-liquid phase separation of DNA nanostructures that leads to the formation of phase-separated capsule-like hydrogels. We designed three types of DNA nanostructures (two orthogonal and a linker nanostructure) that were adsorbed onto an interface of water-in-oil droplets via electrostatic interactions. The phase separation of DNA nanostructures led to the formation of hydrogels of bicontinuous, patch, and mix patterns, due to the immiscibility of liquid-like DNA during the self-assembly process. The frequency of appearance of these patterns was regulated by designing DNA sequences and altering the mixing ratio of the nanostructures. We constructed a phase diagram for the capsule-like DNA hydrogels by investigating pattern formation under various conditions. Our results provide a method for the design and control of phase-separated hydrogel capsules using sequence-designed DNAs. We envision that by incorporating various DNA nanodevices into DNA hydrogel capsules, the capsules will gain molecular sensing, chemical-information processing, and mechano-chemical actuating functions, allowing the construction of functional molecular systems.</p>

scholarly journals Liquid-Liquid Phase Separation of Tau Driven by Hydrophobic Interaction Facilitates Fibrillization of Tau

2021 ◽  
Vol 433 (2) ◽  
pp. 166731
Author(s):  
Yanxian Lin ◽  
Yann Fichou ◽  
Andrew P. Longhini ◽  
Luana C. Llanes ◽  
Pengyi Yin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Hydrophobic Interaction ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

scholarly journals Amyloid Aggregation under the Lens of Liquid–Liquid Phase Separation

2020 ◽  
pp. 368-378
Author(s):  
Yanting Xing ◽  
Aparna Nandakumar ◽  
Aleksandr Kakinen ◽  
Yunxiang Sun ◽  
Thomas P. Davis ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Amyloid Aggregation ◽  
Liquid Liquid Phase Separation

scholarly journals Observation of liquid-liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

2021 ◽  
Author(s):  
Kazuki Murakami ◽  
Shinji Kajimoto ◽  
Daiki Shibata ◽  
Kunisato Kuroi ◽  
Fumihiko Fujii ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Quantitative Evaluation ◽  
Raman Microscopy ◽  
Liquid Phase Separation ◽  
Biological Processes ◽  
Single Droplet ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to...

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