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

Abstract

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.

Minimal RNA self-reproduction discovered from a random pool of oligomers

“…, the association of substrates). 45–47 Alternatively, as demonstrated for a self-reproducing ligase ribozyme, 48 directed evolution with T G ′ as the parent RNA may also identify highly efficient reproduction of oligonucleotides in a constant environment. It was shown that only a slight difference, including two critical mutations, was sufficient to convert the original ligase ribozyme 13 (Fig.…”

“…These limitations may be overcome if strong chemical activation is adopted instead of >p or in environments that periodically experience low pH, high temperatures, or low MgCl 2 concentrations, which destabilize RNA-RNA interactions (e.g., the association of substrates). [45][46][47] Alternatively, as demonstrated for a self-reproducing ligase ribozyme, 48 directed evolution with T G ′ as the parent RNA may also identify highly efficient reproduction of oligonucleotides in a constant environment. It was shown that only a slight difference, including two critical mutations, was sufficient to convert the original ligase ribozyme 13 (Fig.…”

Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

“…For example, moderately acidic pH can protonate the N 1 of adenine and N 3 of cytosine bases, disrupting base pairing and destabilizing RNA duplexes 10 . Coupled with wet/dry cycles or ionic gradients in a thermophoretic setting, this has been shown to promote duplex melting and enable nucleic acid amplification by proteinaceous enzymes 11–14 . Furthermore, highly viscous solvents can slow RNA reannealing sufficiently for long (32-nt) substrates to be ligated 15, 16 .…”

“…Circumventing full duplex dissociation, strand-displacement syntheses can occur on a short RNA duplex by the progressive addition of “invader” oligonucleotides complementary to the non-templating strand 17 , or by the buildup of conformational strain on circular RNA templates 18 . Despite these advances, the scope of RNA-catalysed RNA replication cycles has been limited: either via polymerisation of NTPs on primers flanking a 4-nt region (assisted by denaturants) 19 , or via templated ligation of (up to) three polynucleotide substrate segments 14, 20 . General RNA replication and open-ended evolution requires both the replication of longer sequences able to encode a phenotype and the polymerisation of building blocks short enough to enable free sequence variation.…”

High-Fidelity RNA Copying via 2′,3′-Cyclic Phosphate Ligation

“…A simple way to overcome these issues could be templated ligation, which reduces the number of steps-per-length required to generate an oligonucleotide, and if possible, operating at low magnesium concentration would offer an easier strand separation, particularly when coupled to nonequilibrium microenvironments with salt and pH cycling. 6 Additionally, ligation chain reactions are known to offer exponential replication. 7 The state-of-the-art method for templated ligation makes use of phosphoramidates, such as phosphorimidazolides.…”