RESUMO
Understanding the dynamics of biomolecules in complex environments is crucial for elucidating the effect of condensed and heterogeneous environments on their functional properties. A relevant environment-and one that can also be mimicked easily in vitro-is that of phase-separated droplets. While phase-separated droplet systems have been shown to compartmentalize a wide range of functional biomolecules, the effects of internal structuration of droplets on the dynamics and mobility of internalized molecules remain poorly understood. Here, we use fluorescence correlation spectroscopy to measure the dynamics of short oligonucleotides encapsulated within two representative kinds of uncharged and charged phase-separated droplets. We find that the internal structuration controls the oligonucleotide dynamics in these droplets, revealed by measuring physical parameters at high spatiotemporal resolution. By varying oligonucleotide length and salt concentrations (and thereby charge screening), we found that the dynamics are significantly affected in the noncharged droplets compared to the charged system. Our work lays the foundation for unraveling and quantifying the physical parameters governing biomolecular transport in the condensed environment.
Assuntos
DNA , DNA/química , Oligonucleotídeos/química , Espectrometria de Fluorescência , Oligodesoxirribonucleotídeos/químicaRESUMO
The control of gene expression in cells and organisms allows to unveil gene to function relationships and to reprogram biological responses. Several systems, such as Zinc fingers, TALE (Transcription activator-like effectors), and siRNAs (small-interfering RNAs), have been exploited to achieve this. However, recent advances in Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 (CRISPR-Cas9) have overshadowed them due to high specificity, compatibility with many different organisms, and design flexibility. In this review we summarize state-of-the art for CRISPR-Cas9 technology for large scale gene perturbation studies, including single gene and multiple genes knock-out, knock-down, knock-up libraries, and their associated screening assays. We feature in particular the combination of these methods with single-cell transcriptomics approaches. Finally, we highlight the application of CRISPR-Cas9 systems in building synthetic circuits that can be interfaced with gene networks to control cellular states.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Animais , Redes Reguladoras de Genes/genética , HumanosRESUMO
The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.
Assuntos
RNA Bacteriano/metabolismo , RNA Catalítico/metabolismo , Azoarcus/genética , Azoarcus/metabolismo , Catálise , Regulação Bacteriana da Expressão Gênica , Homeostase , Redes e Vias Metabólicas/genética , Metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/classificação , RNA Catalítico/químicaRESUMO
Catalytic RNAs are attractive objects for studying molecular evolution. To understand how RNA libraries can evolve from randomness toward highly active catalysts, we analyze the original samples that led to the discovery of Diels-Alderase ribozymes by next-generation sequencing. Known structure-activity relationships are used to correlate abundance with catalytic performance. We find that efficient catalysts arose not just from selection for reactivity among the members of the starting library, but from improvement of less potent precursors by mutations. We observe changes in the ribozyme population in response to increasing selection pressure. Surprisingly, even after many rounds of enrichment, the libraries are highly diverse, suggesting that potential catalysts are more abundant in random space than generally thought. To highlight the use of next-generation sequencing as a tool for in vitro selections, we also apply this technique to a recent, less characterized ribozyme selection. Making use of the correlation between sequence evolution and catalytic activity, we predict mutations that improve ribozyme activity and validate them biochemically. Our study reveals principles underlying ribozyme in vitro selections and provides guidelines to render future selections more efficient, as well as to predict the conservation of key structural elements, allowing the rational improvement of catalysts.
Assuntos
RNA Catalítico/química , Evolução Molecular Direcionada , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNARESUMO
Here we report an efficient method for the synthesis of RNA-peptide conjugates by inverse-electron demand Diels-Alder reaction. Various dienophiles were enzymatically incorporated into RNA and reacted with a chemically synthesized diene-modified peptide. The Diels-Alder reaction proceeds with near-quantitative yields in aqueous solution with stoichiometric amounts of reactants, even at low micromolar concentrations.
Assuntos
Reação de Cicloadição , Elétrons , Peptídeos/síntese química , RNA/síntese química , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Nucleotídeos/síntese química , Nucleotídeos/química , Peptídeos/química , RNA/químicaRESUMO
Robust localization of self-reproducing autocatalytic chemistries is a key step in the realization of heritable and evolvable chemical systems. While autocatalytic chemical reaction networks already possess attributes such as heritable self-reproduction and evolvability, localizing functional multispecies networks within complex primitive phases, such as coacervates, has remained unexplored. Here, we show the self-reproduction of the Azoarcus ribozyme system within charge-rich coacervates where catalytic ribozymes are produced by the autocatalytic assembly of constituent smaller RNA fragments. We systematically demonstrate the catalytic assembly of active ribozymes within phase-separated coacervates-both in micron-sized droplets as well as in a coalesced macrophase, underscoring the facility of the complex, charge-rich phase to support these reactions in multiple configurations. By constructing multispecies reaction networks, we show that these newly assembled molecules are active, participating both in self- and cross-catalysis within the coacervates. Finally, due to differential molecular transport, these phase-separated compartments endow robustness to the composition of the collectively autocatalytic networks against external perturbations. Altogether, our results establish the formation of multispecies self-reproducing reaction networks in phase-separated compartments which in turn render transient robustness to the network composition.
RESUMO
Understanding the emergence of life from (primitive) abiotic components has arguably been one of the deepest and yet one of the most elusive scientific questions. Notwithstanding the lack of a clear definition for a living system, it is widely argued that heredity (involving self-reproduction) along with compartmentalization and metabolism are key features that contrast living systems from their non-living counterparts. A minimal living system may be viewed as "a self-sustaining chemical system capable of Darwinian evolution". It has been proposed that autocatalytic sets of chemical reactions (ACSs) could serve as a mechanism to establish chemical compositional identity, heritable self-reproduction, and evolution in a minimal chemical system. Following years of theoretical work, autocatalytic chemical systems have been constructed experimentally using a wide variety of substrates, and most studies, thus far, have focused on the demonstration of chemical self-reproduction under specific conditions. While several recent experimental studies have raised the possibility of carrying out some aspects of experimental evolution using autocatalytic reaction networks, there remain many open challenges. In this review, we start by evaluating theoretical studies of ACSs specifically with a view to establish the conditions required for such chemical systems to exhibit self-reproduction and Darwinian evolution. Then, we follow with an extensive overview of experimental ACS systems and use the theoretically established conditions to critically evaluate these empirical systems for their potential to exhibit Darwinian evolution. We identify various technical and conceptual challenges limiting experimental progress and, finally, conclude with some remarks about open questions.
RESUMO
Discovering autocatalytic chemistries that can evolve is a major goal in systems chemistry and a critical step towards understanding the origin of life. Autocatalytic networks have been discovered in various chemistries, but we lack a general understanding of how network topology controls the Darwinian properties of variation, differential reproduction, and heredity, which are mediated by the chemical composition. Using barcoded sequencing and droplet microfluidics, we establish a landscape of thousands of networks of RNAs that catalyze their own formation from fragments, and derive relationships between network topology and chemical composition. We find that strong variations arise from catalytic innovations perturbing weakly connected networks, and that growth increases with global connectivity. These rules imply trade-offs between reproduction and variation, and between compositional persistence and variation along trajectories of network complexification. Overall, connectivity in reaction networks provides a lever to balance variation (to explore chemical states) with reproduction and heredity (persistence being necessary for selection to act), as required for chemical evolution.
Assuntos
Biocatálise , Redes e Vias Metabólicas , RNA/metabolismoRESUMO
We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.
Assuntos
RNA Bacteriano/genética , RNA Catalítico/genética , RNA/genética , Azoarcus/enzimologia , Biocatálise , Fenômenos Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Sequências Repetidas Invertidas , Cinética , RNA/química , RNA Bacteriano/química , RNA Catalítico/química , Recombinases/química , Recombinases/genéticaRESUMO
We report the first example of RNA labeling based on inverse electron-demand Diels-Alder reactions. Both chemically synthesized and enzymatically transcribed RNAs were successfully modified with biotin or a fluorescent label. This approach works efficiently under mild conditions in water and does not require transition metals.