Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

Nature Nanotechnology, Год журнала: 2025, Номер unknown

Опубликована: Март 17, 2025

Abstract Bottom-up synthetic biology seeks to engineer a cell from molecular building blocks. Using DNA nanotechnology, blocks, such as cytoskeletons, have been reverse-engineered. However, nanostructures rely on chemical synthesis and thermal annealing, therefore cells cannot produce them their constituents nucleotides. Here we introduce RNA origami cytoskeleton mimics alternative nucleic acid-based hardware for cells, which express directly inside giant unilamellar lipid vesicles (GUVs) containing template polymerase, chemically fuelled by feeding nucleotides the outside. We designed tiles that fold upon transcription self-assemble into micrometre-long, three-dimensional nanotubes under isothermal conditions. observe sequence mutations lead closed-ring phenotypes. Molecular dynamics simulations show these phenotypic transitions are governed alterations in stability of secondary structures. In addition, achieve cortex formation with aptamer-functionalized nanotube polymerization leads membrane deformation. Altogether, our data suggest expression origami-based will help explore active, evolvable RNA-based cells.

Язык: Английский

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Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes

International Journal of Molecular Sciences, Год журнала: 2023, Номер 24(8), С. 7231 - 7231

Опубликована: Апрель 13, 2023

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural functional studies of these are essential fields fundamental biology, medical science, pharmacology, biotechnology, bioengineering. However, observing the precise elemental reactions structures membrane is difficult, despite their functioning through interactions with various biomolecules living cells. To investigate properties, methodologies have been developed to study functions that purified from biological In this paper, we introduce methods for creating liposomes or lipid vesicles, conventional recent approaches, well techniques reconstituting into artificial membranes. We also cover different types membranes can be used observe reconstituted proteins, including structure, number transmembrane domains, type. Finally, discuss reconstitution using a cell-free synthesis system function multiple proteins.

Язык: Английский

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Dynamics of giant vesicle assembly from thin lipid films

Journal of Colloid and Interface Science, Год журнала: 2024, Номер 661, С. 1033 - 1045

Опубликована: Фев. 5, 2024

Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution sizes GUVs obtained from film hydration methods were recently reported. Dynamic data such as the time evolution sizes, however, is not known. can provide insights into assembly pathway guidelines for choosing conditions to obtain populations with desired distributions. We develop 'stopped-time' technique characterize molar free-floating GUVs. additionally capture high resolution time-lapse images surface-attached GUV buds on films. systematically study dynamics three widely used methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation aqUeous Solutions), gentle hydration, electroformation. find that yield versus curves demonstrate a characteristic sigmoidal shape, an initial yield, transient, then steady state plateau all methods. The population showed right-skewed diameters. variance distributions increased time. systems reached within 120 min. rationalize using thermodynamically motivated budding merging (BNM) model. These results further understanding first-time practical parameters tailor production specific applications.

Язык: Английский

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Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA

Cell Reports, Год журнала: 2024, Номер 43(4), С. 114084 - 114084

Опубликована: Апрель 1, 2024

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator eosinophil function against pathogens eosinophil-associated diseases. While MBP-1 targets both microorganisms cells, its precise mechanism remains elusive. We demonstrate that formation small pores lipid bilayers induces membrane permeabilization disrupts potassium balance. Additionally, we reveal mitochondrial DNA (mtDNA) present traps (EETs) amplifies toxic effects, underscoring pivotal mtDNA EETs. Furthermore, evidence indicating absence CpG methylation contributes regulation MBP-1-mediated toxicity. Taken together, our data suggest scaffold within promotes

Язык: Английский

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Dissecting Structural Requirements of Leucinostatin A Derivatives for Antiprotozoal Activity and Mammalian Toxicity

Journal of Medicinal Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 14, 2025

Lefleuganan, a clinical stage drug candidate for the treatment of cutaneous leishmaniasis, is synthetic nonapeptide inspired by natural antimicrobial peptide leucinostatin A, exhibiting excellent antiprotozoal activity in low nM range. Lefleuganan shows strongly reduced acute toxicity, making it amenable use. Here, using broad set vivo and vitro measurements purified enzymes, we find that but not lefleuganan, specifically targets mitochondrial ATP synthase, inhibiting synthesis human, bovine, yeast enzyme nanomolar In structure-activity relationship study covering chemical space between two compounds, hydroxyleucine at position 7 A identified as key responsible moiety specific synthase inhibition systemic toxicity. Our data suggest efficient these class compounds mediated energetic uncoupling negatively charged membranes.

Язык: Английский

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Molecular Mechanisms of Action of Dendrimers with Antibacterial Activities on Model Lipid Membranes

Polymers, Год журнала: 2025, Номер 17(7), С. 929 - 929

Опубликована: Март 29, 2025

In the last decades, numerous dendrimers with a variety of potential biomedical applications have been developed and investigated. The aim present study was to evaluate molecular mechanisms interaction between two proven antibacterial activity (4-N,N-dimethylamino-1,8-naphthalimide (Dab) 3-bromo-Dab (Dab-Br)) POPC (1-palmitoyl-2-oleoylphosphatidylcholine) model membranes (monolayers liposomes). pressure-area isotherms compressional modulus monolayers revealed that Dab is likely penetrate hydrophobic region POPC, whereas Dab-Br inserts mainly into lipid headgroup area. This assumption confirmed by FTIR-ATR liposomes containing dendrimers. addition, induced higher order in large unilamellar vesicles (LUVs) compared Dab-Br. Moreover, both changed negative zeta positive values, slightly effect Dab-Br, indicating electrostatic interactions headgroups Furthermore, able reduce average LUVs' size, unlike visualization giant increasing dendrimer concentration membrane shrinking complete disintegration, which more prominent for Dab. Based on experimental results, new fundamental knowledge about destabilizing will be acquired focus their application pharmacology clinical practice.

Язык: Английский

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Electroformation of giant unilamellar vesicles from large liposomes

The European Physical Journal Special Topics, Год журнала: 2024, Номер unknown

Опубликована: Фев. 19, 2024

Abstract Giant unilamellar vesicles (GUVs) are widely used as model systems for biological membranes to study membrane-related processes in a precisely controlled vitro environment, owing their biophysical properties. The classical technique the formation of giant liposomes starts with dissolution lipids or lipid mixtures an organic solvent, which is then deposited thin solution film on support substrate. In this study, we present comprehensive analysis investigating effect different compositions generation GUVs from preformed under non-ionic and ionic conditions. For all liposome types tested, electroformation process, whether performed indium tin oxide-coated glass slides platinum wires, consistently produced that typically ranged size 5 20 μm. However, yield varied depending specific conditions composition used. general, resulting population was predominantly characterised by presence multivesicular vesicles. These findings have potential improve refinement protocol parameters containing membrane proteins effects protein activity.

Язык: Английский

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Genetic encoding and expression of RNA origami cytoskeletons in synthetic cells

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июнь 12, 2024

Abstract The central dogma at the core of molecular biology states that information flows from DNA to RNA and then protein. Our research seeks introduce a conceptually novel approach towards synthetic life by leveraging origami, as an alternative proteins, requiring only single copying step between genetic function. Here, we report encoding expression origami cytoskeleton-mimic within giant unilamellar lipid vesicles (GUVs). We design first tiles which fold co-transcriptionally template self-assemble into higher-order 3D nanotubes constant 37 ◦ C in GUVs, where they reach several micrometers length. Unlike pre-formed encapsulated cytoskeletons, these GUVs produce their own hardware out-of-equilibrium process fuelled nucleotide feeding. To establish genotype-phenotype correlations, investigate how sequence mutations govern contour persistence length with experiments coarse-grained molecular-dynamics simulations, realizing phenotypic transition closed rings. Finally, achieve cortex formation GUV deformation without chemical functionalization introducing aptamers tile design.Altogether, this work pioneers origami-based new active, evolvable RNA-based cells.

Язык: Английский

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Effect of Simulated Microgravity on Artificial Single Cell Membrane Mechanics

Microgravity Science and Technology, Год журнала: 2024, Номер 36(4)

Опубликована: Авг. 13, 2024

Язык: Английский

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Preparation of hydrogel-supported giant vesicles via a lipid-coated hydrogel transfer method with electrostatic interaction

Colloids and Surfaces A Physicochemical and Engineering Aspects, Год журнала: 2024, Номер unknown, С. 136074 - 136074

Опубликована: Дек. 1, 2024

Язык: Английский

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