Periodic temperature changes drive the proliferation of self-replicating RNAs in vesicle populations.

Growth and division of biological cells are based on the complex orchestration of spatiotemporally controlled reactions driven by highly evolved proteins. In contrast, it remains unknown how their primordial predecessors could achieve a stable inheritance of cytosolic components before the advent of translation. An attractive scenario assumes that periodic changes of environmental conditions acted as pacemakers for the proliferation of early protocells. Using catalytic RNA (ribozymes) as models for primitive biocatalytic molecules, we demonstrate that the repeated freezing and thawing of aqueous solutions enables the assembly of active ribozymes from inactive precursors encapsulated in separate lipid vesicle populations. Furthermore, we show that encapsulated ribozyme replicators can overcome freezing-induced content loss and successive dilution by freeze-thaw driven propagation in feedstock vesicles. Thus, cyclic freezing and melting of aqueous solvents - a plausible physicochemical driver likely present on early Earth - provides a simple scenario that uncouples compartment growth and division from RNA self-replication, while maintaining the propagation of these replicators inside new vesicle populations.

Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

Ribozyme-mediated RNA synthesis and replication in a model Hadean microenvironment.

Enzyme-catalyzed replication of nucleic acid sequences is a prerequisite for the survival and evolution of biological entities. Before the advent of protein synthesis, genetic information was most likely stored in and replicated by RNA. However, experimental systems for sustained RNA-dependent RNA-replication are difficult to realise, in part due to the high thermodynamic stability of duplex products and the low chemical stability of catalytic RNAs. Using a derivative of a group I intron as a model for an RNA replicase, we show that heated air-water interfaces that are exposed to a plausible CO2-rich atmosphere enable sense and antisense RNA replication as well as template-dependent synthesis and catalysis of a functional ribozyme in a one-pot reaction. Both reactions are driven by autonomous oscillations in salt concentrations and pH, resulting from precipitation of acidified dew droplets, which transiently destabilise RNA duplexes. Our results suggest that an abundant Hadean microenvironment may have promoted both replication and synthesis of functional RNAs.

CRISPR Interference Modules as Low-Burden Logic Inverters in Synthetic Circuits.

CRISPR and CRISPRi systems have revolutionized our biological engineering capabilities by enabling the editing and regulation of virtually any gene, via customization of single guide RNA (sgRNA) sequences. CRISPRi modules can work as programmable logic inverters, in which the dCas9-sgRNA complex represses a target transcriptional unit. They have been successfully used in bacterial synthetic biology to engineer information processing tasks, as an alternative to the traditionally adopted transcriptional regulators. In this work, we investigated and modulated the transfer function of several model systems with specific focus on the cell load caused by the CRISPRi logic inverters. First, an optimal expression cassette for dCas9 was rationally designed to meet the low-burden high-repression trade-off. Then, a circuit collection was studied at varying levels of dCas9 and sgRNAs targeting three different promoters from the popular tet, lac and lux systems, placed at different DNA copy numbers. The CRISPRi NOT gates showed low-burden properties that were exploited to fix a high resource-consuming circuit previously exhibiting a non-functional input-output characteristic, and were also adopted to upgrade a transcriptional regulator-based NOT gate into a 2-input NOR gate. The obtained data demonstrate that CRISPRi-based modules can effectively act as low-burden components in different synthetic circuits for information processing.

Nucleic Acid Catalysis under Potential Prebiotic Conditions.

Catalysis by nucleic acids is indispensable for extant cellular life, and it is widely accepted that nucleic acid enzymes were crucial for the emergence of primitive life 3.5-4 billion years ago. However, geochemical conditions on early Earth must have differed greatly from the constant internal milieus of today's cells. In order to explore plausible scenarios for early molecular evolution, it is therefore essential to understand how different physicochemical parameters, such as temperature, pH, and ionic composition, influence nucleic acid catalysis and to explore to what extent nucleic acid enzymes can adapt to non-physiological conditions. In this article, we give an overview of the research on catalysis of nucleic acids, in particular catalytic RNAs (ribozymes) and DNAs (deoxyribozymes), under extreme and/or unusual conditions that may relate to prebiotic environments.

Active coacervate droplets as a model for membraneless organelles and protocells.

Membraneless organelles like stress granules are active liquid-liquid phase-separated droplets that are involved in many intracellular processes. Their active and dynamic behavior is often regulated by ATP-dependent reactions. However, how exactly membraneless organelles control their dynamic composition remains poorly understood. Herein, we present a model for membraneless organelles based on RNA-containing active coacervate droplets regulated by a fuel-driven reaction cycle. These droplets emerge when fuel is present, but decay without. Moreover, we find these droplets can transiently up-concentrate functional RNA which remains in its active folded state inside the droplets. Finally, we show that in their pathway towards decay, these droplets break apart in multiple droplet fragments. Emergence, decay, rapid exchange of building blocks, and functionality are all hallmarks of membrane-less organelles, and we believe that our work could be powerful as a model to study such organelles.

Overview of the Formulations and Analogs in the Taxanes' Story.

Taxanes are a family of diterpenes produced by the yews (Taxus genus) that are extensively used in chemotherapy. The family encompasses paclitaxel, docetaxel and the recently added cabazitaxel, all of which were proven to be promising anti-cancer drugs. Due to the over harvesting danger threatening the yew trees as well as the many challenges faced by taxane-based chemotherapy, new formulations, analogs and delivery systems are required. Here, we undertook a structured search of the bibliographical database PubMed for peerreviewed research papers relying on key words and date of publication and organized the information based on the method of taxane drug delivery. Papers retrieved were from journals with significant impact and comparable scope. A total of 126 papers were reviewed, 81 of which published work related to the taxane formulations and nanoparticles, and 22 focused on the analogs derived from the three taxanes. Although recent articles investigate the effectiveness of taxane formulations, most of these formulations are still at the pre-clinical level. However, many of the taxane analogs are currently in clinical trials as second line treatment of aggressive cancers or are used in combination with other chemotherapeutic drugs. The findings corroborate the importance of developing new drug delivery strategies and taxane analogs to improve the efficacy of currently used chemotherapeutic drugs. This finding is further supported by the FDA-approved formulation of paclitaxel that eliminates the need for toxic solvents for drug administration, and the docetaxel analog cabazitaxel which has decreased affinity for efflux pumps.