Efficient enzyme-free copying of all four nucleobases templated by immobilized RNA

Deck, Christopher; Jauker, Mario; Richert, Clemens

doi:10.1038/nchem.1086

Cited by 133 publications

(179 citation statements)

References 28 publications

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“…In one approach to strand inhibition, template strands are immobilized on a solid particle support while undergoing cycles where complementary fragments are fed, ligated, washed, and product strands re-immobilized; this process resulted in desirable "exponential" replication (doubling of the template population with every round) [123]. In a similar system, additional binding of downstream "micro-helper" oligomers to the template strand allowed for efficient copying of all four RNA nucleobases [124]. Though not a self-sustained system, these studies emphasize the potential importance of solid-phase chemistry and periodic replacement of monomers/fragments in driving replication processes.…”

Section: Selectionmentioning

confidence: 99%

A Chemical Engineering Perspective on the Origins of Life

Grover

Hsieh

et al. 2015

Processes

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Atoms and molecules assemble into materials, with the material structure determining the properties and ultimate function. Human-made materials and systems have achieved great complexity, such as the integrated circuit and the modern airplane. However, they still do not rival the adaptivity and robustness of biological systems. Understanding the reaction and assembly of molecules on the early Earth is a scientific grand challenge, and also can elucidate the design principles underlying biological materials and systems. This research requires understanding of chemical reactions, thermodynamics, fluid mechanics, heat and mass transfer, optimization, and control. Thus, the discipline of chemical engineering can play a central role in advancing the field. In this paper, an overview of research in the origins field is given, with particular emphasis on the origin of biopolymers and the role of chemical engineering phenomena. A case study is presented to highlight the importance of the environment and its coupling to the chemistry.

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Section: Selectionmentioning

confidence: 99%

A Chemical Engineering Perspective on the Origins of Life

Grover

Hsieh

et al. 2015

Processes

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“…Early pioneering approaches are reviewed in [21][22][23]. Efficient enzyme-free copying of all four pre-activated nucleobases, templated by immobilized RNA, was reported [24].…”

Section: Introductionmentioning

confidence: 99%

Ribozyme Activity of RNA Nonenzymatically Polymerized from 3′,5′-Cyclic GMP

Pino

Costanzo

Giorgi

et al. 2013

Entropy

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3′,5′-Cyclic GMP spontaneously nonenzymatically polymerizes in a base-catalyzed reaction affording G oligonucleotides. When reacted with fully or partially sequence-complementary RNA (oligo C), the abiotically generated oligo G RNA displays a typical ribozyme activity consisting of terminal ligation accompanied by cleavage of an internal phosphate site of the donor oligonucleotide stem upon attack of the acceptor 3′ terminal OH. This reaction is dubbed Ligation following Intermolecular Cleavage (LIC). In a prebiotic perspective, the ability of oligo G polynucleotides to react with other sequences outlines a simple and possible evolutionary scenario based on the autocatalytic properties of RNA.

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“…The efficiency of non-enzymatic RNA replication can be greatly enhanced by the periodic addition of fresh portions of activated monomer to a primer-extension reaction occurring on templates immobilized by covalent linkage to beads (8). We sought to reproduce this effect by mimicking the flow of an external solution of fresh monomers over vesicles, by periodic dialysis of model protocells against a solution of fresh activated monomers (See Supporting Materials for description of the Liposome Reactor Dialyzer).…”

mentioning

confidence: 99%

Nonenzymatic Template-Directed RNA Synthesis Inside Model Protocells

Adamala

Szostak

2013

Science

296

307

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Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg 2+ on fatty acid membranes. Here, we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg 2+ ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg 2+ -catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.The RNA world hypothesis suggests that the primordial catalysts were ribozymes (1, 2), while biophysical considerations suggest that the primordial replicating compartments were membranous vesicles composed of fatty acids and related amphiphiles (3, 4). However, the conditions required for RNA replication chemistry and fatty acid vesicle integrity have appeared to be fundamentally incompatible (5) (Fig S1). Both ribozyme catalyzed and nonenzymatic RNA copying reactions require high (50-200 mM) concentrations of Mg 2+ (or other divalent) ions (6), but Mg 2+ at such concentrations destroys vesicles by causing fatty acid precipitation.We developed a screen for small molecules that protect oleate fatty acid vesicles from disruption by Mg 2+ . We used two assays to monitor the leakage of either a small charged molecule (calcein) or a larger oligonucleotide, allowing us to distinguish between increased (Fig. S5 and S6). In the presence of chelated Mg 2+ , oleate vesicles remained intact but exhibited a modest increase in the permeability of a small polar molecule ( Fig. 1 and S7) and an even smaller increase in the leakage of an oligonucleotide. In terms of vesicle stabilization, citrate was one of the most effective chelators of Mg 2+ .We also examined the stability of model protocell membranes made of myristoleic acid: glycerol monomyristoleate 2:1 and from the more prebiotically reasonable decanoic acid: decanol: glycerol monodecanoate 4:1:1. Citrate-chelated Mg 2+ caused only a small amount of leakage from these vesicles, and the stabilizing effect of citrate was seen for both calcein and oligonucleotides ( Fig. 1 and S8-S13).We then asked if these chelators were compatible with the Mg 2+ catalysis of non-enzymatic template-directed RNA primer extension. We measured the rate at which an RNA primer was elongated when annealed to an oligonucleotide with a templating region of C nucleotides, in the presence of excess activated G monomer, guanosine 5′-phosphor(2-methyl)imidazolide (2MeImpG) (Fig. 2). We examined citric acid, EDTA, NTA and a weakly stabilizing chelator (isocitric acid). In the presence of 50 mM unchelated Mg 2+ , the primer-extension reaction proceeded at a rate of 1.4 h 1 , compared to 0.03 h −1 in the absence of Mg 2+ ions. The addition of 4 equivalents of EDTA or NTA resulted in co...

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Efficient enzyme-free copying of all four nucleobases templated by immobilized RNA

Cited by 133 publications

References 28 publications

A Chemical Engineering Perspective on the Origins of Life

A Chemical Engineering Perspective on the Origins of Life

Ribozyme Activity of RNA Nonenzymatically Polymerized from 3′,5′-Cyclic GMP

Nonenzymatic Template-Directed RNA Synthesis Inside Model Protocells

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