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.

PCR Methods for the Generation of Catalytic ssDNA.

The ability to produce single-stranded DNA on a preparative scale from low amounts of starting templates is necessary for most research involving deoxyribozymes, but is particularly important for performing in vitro selections. While the production of single-stranded RNA is straightforward by means of in vitro transcription, the enzymatic production of single-stranded DNA (ssDNA) on a preparative scale is often difficult. Nevertheless, several methods for the production of ssDNA have been published over the years. Here, we present two PCR methods that we find to be particularly effective, fast, and affordable, which we have adapted for our own needs.

Generation of RNA with 2', 3'-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions.

The generation of terminal 2', 3'-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2', 3'-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3'-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Prebiotically Plausible RNA Activation Compatible with Ribozyme-Catalyzed Ligation.

RNA-catalyzed RNA ligation is widely believed to be a key reaction for primordial biology. However, since typical chemical routes towards activating RNA substrates are incompatible with ribozyme catalysis, it remains unclear how prebiotic systems generated and sustained pools of activated building blocks needed to form increasingly larger and complex RNA. Herein, we demonstrate in situ activation of RNA substrates under reaction conditions amenable to catalysis by the hairpin ribozyme. We found that diamidophosphate (DAP) and imidazole drive the formation of 2',3'-cyclic phosphate RNA mono- and oligonucleotides from monophosphorylated precursors in frozen water-ice. This long-lived activation enables iterative enzymatic assembly of long RNAs. Our results provide a plausible scenario for the generation of higher-energy substrates required to fuel ribozyme-catalyzed RNA synthesis in the absence of a highly evolved metabolism.

Controlling Protein Nanocage Assembly with Hydrostatic Pressure.

Controlling the assembly and disassembly of nanoscale protein cages for the capture and internalization of protein or non-proteinaceous components is fundamentally important to a diverse range of bionanotechnological applications. Here, we study the reversible, pressure-induced dissociation of a natural protein nanocage, E. coli bacterioferritin (Bfr), using synchrotron radiation small-angle X-ray scattering (SAXS) and circular dichroism (CD). We demonstrate that hydrostatic pressures of 450 MPa are sufficient to completely dissociate the Bfr 24-mer into protein dimers, and the reversibility and kinetics of the reassembly process can be controlled by selecting appropriate buffer conditions. We also demonstrate that the heme B prosthetic group present at the subunit dimer interface influences the stability and pressure lability of the cage, despite its location being discrete from the interdimer interface that is key to cage assembly. This indicates a major cage-stabilizing role for heme within this family of ferritins.

The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1-17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all ß-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

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.

Designer artificial membrane binding proteins to direct stem cells to the myocardium.

We present a new cell membrane modification methodology where the inherent heart tissue homing properties of the infectious bacteria Streptococcus gordonii are transferred to human stem cells. This is achieved via the rational design of a chimeric protein-polymer surfactant cell membrane binding construct, comprising the cardiac fibronectin (Fn) binding domain of the bacterial adhesin protein CshA fused to a supercharged protein. Significantly, the protein-polymer surfactant hybrid spontaneously inserts into the plasma membrane of stem cells without cytotoxicity, instilling the cells with a high affinity for immobilized fibronectin. Moreover, we show that this cell membrane reengineering approach significantly improves retention and homing of stem cells delivered either intracardially or intravenously to the myocardium in a mouse model.

Heated gas bubbles enrich, crystallize, dry, phosphorylate and encapsulate prebiotic molecules.

Non-equilibrium conditions must have been crucial for the assembly of the first informational polymers of early life, by supporting their formation and continuous enrichment in a long-lasting environment. Here, we explore how gas bubbles in water subjected to a thermal gradient, a likely scenario within crustal mafic rocks on the early Earth, drive a complex, continuous enrichment of prebiotic molecules. RNA precursors, monomers, active ribozymes, oligonucleotides and lipids are shown to (1) cycle between dry and wet states, enabling the central step of RNA phosphorylation, (2) accumulate at the gas-water interface to drastically increase ribozymatic activity, (3) condense into hydrogels, (4) form pure crystals and (5) encapsulate into protecting vesicle aggregates that subsequently undergo fission. These effects occur within less than 30 min. The findings unite, in one location, the physical conditions that were crucial for the chemical emergence of biopolymers. They suggest that heated microbubbles could have hosted the first cycles of molecular evolution.

The difficult case of an RNA-only origin of life.

The RNA world hypothesis is probably the most extensively studied model for the emergence of life on Earth. Despite a large body of evidence supporting the idea that RNA is capable of kick-starting autocatalytic self-replication and thus initiating the emergence of life, seemingly insurmountable weaknesses in the theory have also been highlighted. These problems could be overcome by novel experimental approaches, including out-of-equilibrium environments, and the exploration of an early co-evolution of RNA and other key biomolecules such as peptides and DNA, which might be necessary to mitigate the shortcomings of RNA-only systems.

Templated Self-Replication in Biomimetic Systems.

A key characteristic of living systems is the storage and replication of information, and as such the development of self-replicating systems capable of heredity is of great importance to the fields of synthetic biology and origin of life research. In this review, the design and implementation of self-replicating systems in the context of bottom-up synthetic biology is discussed, with a particular focus on nucleic acid-based replication including nonenzymatic systems, ribozyme-based systems, and complex in vitro translation coupled RNA and DNA replication. The current state and remaining challenges of the respective fields are discussed, and the potential of individual replicators for synthetic biology applications such as the creation of artificial life capable of Darwinian evolution is also summarized.

Compartmentalised RNA catalysis in membrane-free coacervate protocells.

Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free droplets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2.

Functionalized Triblock Copolymer Vectors for the Treatment of Acute Lymphoblastic Leukemia.

The chemotherapeutic Parthenolide is an exciting new candidate for the treatment of acute lymphoblastic leukemia, but like many other small-molecule drugs, it has low aqueous solubility. As a consequence, Parthenolide can only be administered clinically in the presence of harmful cosolvents. Accordingly, we describe the synthesis, characterization, and testing of a range of biocompatible triblock copolymer micelles as particle-based delivery vectors for the hydrophobic drug Parthenolide. The drug-loaded particles are produced via an emulsion-to-micelle transition method, and the effects of introducing anionic and cationic surface charges on stability, drug sequestration, biocompatibility, and efficacy are investigated. Significantly, we demonstrate high levels of efficacy in the organic solvent-free systems against human mesenchymal stem cells and primary T-acute lymphoblastic leukemia patient cells, highlighting the effectiveness of the delivery vectors for the treatment of acute lymphoblastic leukemia.