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Szostak, J. W.

Publications and source records attributed to Szostak, J. W..

41 records · Page 3Linked to original sources

A model for the emergence of RNA from a prebiotically plausible mixture of ribonucleotides, arabinonucleotides and 2’-deoxynucleotides

The abiotic synthesis of ribonucleotides is thought to have been an essential step towards the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabino-nucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabino-nucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

biochemistry↗

Non-enzymatic primer extension with strand displacement

Non-enzymatic RNA self-replication is integral to the RNA World hypothesis. Despite considerable progress in non-enzymatic template copying, true replication remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible solution to this problem in which short invader oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3'-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been catalyzed by short RNA oligonucleotides.

biochemistry↗

Prebiotically Plausible ‘Patching’ of RNA Backbone Cleavage Through a 3′-5′ Pyrophosphate Linkage

Achieving multiple cycles of RNA replication within a model protocell would be a critical step towards demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an RNA world must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an endpoint for RNA replication. Instead, 3' -phosphate terminated RNA strands are able to participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. We found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. Under these conditions, pyrophosphate-RNA can act as a temporary patch to template the polymerization of canonical ribonucleotides, suggesting a plausible non-enzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

biochemistry↗

Interpreting turbidity measurements for vesicle studies

Spectrophotometers are routinely used to assess the turbidity of vesicle solutions. Here we provide guidelines for interpreting turbidity measurements of vesicle samples, and highlight potential pitfalls of the approach. We use an exact solution for core-shell scatterers to model and calculate how samples of vesicles scatter light, and provide a comprehensive overview of how the turbidity of vesicle samples can change with vesicle size, contents, and composition. Surprisingly, we find that vesicle lamellarity has a large effect on sample turbidity, while unilamellar vesicles of different sizes have similar turbidity. We use our model in conjunction with experimental data to measure the thickness of oleic acid vesicle membranes and find excellent agreement with values determined by cryo-TEM. We also calculate the effects of potential errors in measurement from forward scattering and multiple scattering.

biophysics↗

Crystallographic observation of nonenzymatic RNA primer extension

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

biochemistry↗