168 research outputs found
RNA catalysis in model protocell vesicles.
We are engaged in a long-term effort to synthesize chemical systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and divide, but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes, because low concentrations of divalent cations (e.g., Mg(2+)) cause fatty acids to precipitate. Furthermore, vesicles that grow and divide must be permeable to the cations and substrates required for internal metabolism. We used a mixture of myristoleic acid and its glycerol monoester to construct vesicles that were Mg(2+)-tolerant and found that Mg(2+) cations can permeate the membrane and equilibrate within a few minutes. In vesicles encapsulating a hammerhead ribozyme, the addition of external Mg(2+) led to the activation and self-cleavage of the ribozyme molecules. Vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles, and further modification of the membrane composition allowed growth following mixed micelle addition. Our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg(2+) and mononucleotides without specific macromolecular transporters. This combination of stability and dynamics is critical for building model protocells in the laboratory and may have been important for early cellular evolution
Isolation of a fluorophore-specific DNA aptamer with weak redox activity
AbstractBackground: In vitro selection experiments with pools of random-sequence nucleic acids have been used extensively to isolate molecules capable of binding specific ligands and catalyzing self-modification reactions.Results: In vitro selection from a random pool of single-stranded DNAs has been used to isolate molecules capable of recognizing the fluorophore sulforhodamine B with high affinity. When assayed for the ability to promote an oxidation reaction using the reduced form of a related fluorophore, dihydrotetramethylrosamine, a number of selected clones show low levels of catalytic activity. Chemical modification and site-directed mutagenesis experiments have been used to probe the structural requirements for fluorophore binding. The aptamer recognizes its ligand with relatively high affinity and is also capable of binding related molecules that share extended aromatic rings and negatively charged functional groups.Conclusions: A guanosine-rich single-stranded DNA is capable of binding fluorophores with relatively high affinity and of weakly promoting a multiple-turnover reaction. A simple motif consisting of a three-tiered G-quartet stacked upon a standard Watson-Crick duplex appears to be responsible for this activity. The corresponding sequence might provide a useful starting point for the evolution of novel, improved deoxyribozymes that generate fluorescent signals by promoting multiple-turnover reactions
Isolation of novel ribozymes that ligate AMP-activated RNA substrates
AbstractBackground: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5′-5′-pyrophosphate ‘capped’ RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5′-monophosphate (AMP) may be a vestige of ‘RNA world’ catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.Results: We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 1015 RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5′-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of ∼ 5 × 105 over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3′-5′-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.Conclusions: The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates
The Emergence of Competition Between Model Protocells
The transition from independent molecular entities to cellular structures with
integrated behaviors was a crucial aspect of the origin of life. We show that simple
physical principles can mediate a coordinated interaction between genome and
compartment boundary, independent of any genomic functions beyond self-replication.
RNA, encapsulated in fatty acid vesicles, exerts an osmotic pressure on
the vesicle membrane that drives the uptake of additional membrane components,
leading to membrane growth at the expense of relaxed vesicles, which shrink. Thus,
more efficient RNA replication could cause faster cell growth, leading to the
emergence of Darwinian evolution at the cellular level
An optimal degree of physical and chemical heterogeneity for the origin of life?
The accumulation of pure, concentrated chemical building blocks, from which the essential components of protocells could be assembled, has long been viewed as a necessary, but extremely difficult step on the pathway to the origin of life. However, recent experiments have shown that moderately increasing the complexity of a set of chemical inputs can in some cases lead to a dramatic simplification of the resulting reaction products. Similarly, model protocell membranes composed of certain mixtures of amphiphilic molecules have superior physical properties than membranes composed of single amphiphiles. Moreover, membrane self-assembly under simple and natural conditions gives rise to heterogeneous mixtures of large multi-lamellar vesicles, which are predisposed to a robust pathway of growth and division that simpler and more homogeneous small unilamellar vesicles cannot undergo. Might a similar relaxation of the constraints on building block purity and homogeneity actually facilitate the difficult process of nucleic acid replication? Several arguments suggest that mixtures of monomers and short oligonucleotides may enable the chemical copying of polynucleotides of sufficient length and sequence complexity to allow for the emergence of the first nucleic acid catalysts. The question of the origin of life may become less daunting once the constraints of overly well-defined laboratory experiments are appropriately relaxed
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Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets
Compartmentalization in a prebiotic setting is an important aspect of early cell formation and is crucial for the development of an artificial protocell system that effectively couples genotype and phenotype. Aqueous two-phase systems (ATPSs) and complex coacervates are phase separation phenomena that lead to the selective partitioning of biomolecules and have recently been proposed as membrane-free protocell models. We show in this study through fluorescence recovery after photobleaching (FRAP) microscopy that despite the ability of such systems to effectively concentrate RNA, there is a high rate of RNA exchange between phases in dextran/polyethylene glycol ATPS and ATP/poly-L-lysine coacervate droplets. In contrast to fatty acid vesicles, these systems would not allow effective segregation and consequent evolution of RNA, thus rendering these systems ineffective as model protocells. Electronic supplementary material The online version of this article (doi:10.1007/s11084-014-9355-8) contains supplementary material, which is available to authorized users
Streptavidin-binding peptides and uses thereof
The invention provides peptides with high affinity for streptavidin. These peptides may be expressed as part of fusion proteins to facilitate the detection, quantitation, and purification of proteins of interest
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Multicomponent Assembly of Proposed DNA Precursors in Water
We propose a novel pathway for the prebiotic synthesis of 2′-deoxynucleotides. Consideration of the constitutional chemical relationships between glycolaldehyde and β-mercapto-acetaldehyde, and the corresponding proteinogenic amino acids, serine and cysteine, led us to explore the consequences of the corresponding sulfur substitution for our previously proposed pathways leading to the canonical ribonucleotides. We demonstrate that just as 2-aminooxazole–an important prebiotic ribonucleotide precursor–is readily formed from glycolaldehyde and cyanamide, so is 2-aminothiazole formed from β-mercapto-acetaldehyde and cyanamide in water at neutral pH. Indeed, both the oxazole and the thiazole can be formed together in a one-pot reaction, and can be co-purified by crystallization or sublimation. We then show that 2-aminothiazole can take part in a 3-component carbon–carbon bond-forming reaction in water that leads to the diastereoselective synthesis of masked 2′-thiosugars regiospecifically tethered to purine precursors, which would lead to 2′-deoxynucleotides upon desulfurization. The possibility of an abiotic route to the 2′-deoxynucleotides provides a new perspective on the evolutionary origins of DNA. We also show that 2-aminothiazole is able to sequester, through reversible aminal formation, the important nucleotide precursors glycolaldehyde and glyceraldehyde in a stable, crystalline form
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Large Phenotypic Enhancement of Structured Random RNA Pools.
Laboratory evolution of functional RNAs has applications in many areas of chemical and synthetic biology. In vitro selections critically depend on the presence of functional molecules, such as aptamers and ribozymes, in the starting sequence pools. For selection of novel functions the pools are typically transcribed from random-sequence DNA templates, yielding a highly diverse set of RNAs that contain a multitude of folds and biochemical activities. The phenotypic potential, the frequency of functional RNAs, is very low, requiring large complexity of starting pools, surpassing 1015 different sequences, to identify highly active isolates. Furthermore, the majority of random sequences is not structured and has a high propensity for aggregation; the in vitro selection process thus involves not just enrichment of functional RNAs, but also their purification from aggregation-prone "free-riders". We reasoned that purification of the nonaggregating, monomeric subpopulation of a random-sequence RNA pool will yield pools of folded, functional RNAs. We performed six rounds of selection for monomeric sequences and show that the enriched population is compactly folded. In vitro selections originating from various mixtures of the compact pool and a fully random pool showed that sequences from the compact pool always dominate the population once a biochemical activity is detectable. A head-to-head competition of the two pools starting from a low (5 × 1012) sequence diversity revealed that the phenotypic potential of the compact pool is about 1000-times higher than the fully random pool. A selection for folded and monomeric RNA pools thus greatly increases the frequency of functional RNAs from that seen in random-sequence pools, providing a facile experimental approach to isolation of highly active functional RNAs from low-diversity populations
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