Lower temperature optimum of a smaller, fragmented triphosphorylation ribozyme

Abstract

The RNA world hypothesis describes a stage in the early evolution of life in which catalytic RNAs mediated the replication of RNA world organisms. One challenge to this hypothesis is that most existing ribozymes are much longer than what may be expected to originate from prebiotically plausible methods, or from the polymerization by currently existing polymerase ribozymes. We previously developed a 96-nucleotide long ribozyme, which generates a chemically activated 5'-phosphate (a 5'-triphosphate) from a prebiotically plausible molecule, trimetaphosphate, and an RNA 5'-hydroxyl group. Analogous ribozymes may have been important in the RNA world to access an energy source for the earliest life forms. Here we reduce the length of this ribozyme by fragmenting the ribozyme into multiple RNA strands, and by successively removing its longest double strand. The resulting ribozyme is composed of RNA fragments with none longer than 34 nucleotides. The temperature optimum was ∼20 °C, compared to ∼40 °C for the parent ribozyme. This shift in temperature dependence may be a more general phenomenon for fragmented ribozymes, and may have helped RNA world organisms to emerge at low temperature.

A combinatorial method to isolate short ribozymes from complex ribozyme libraries

“…The pH dependence of the 44-nucleotide ribozyme shows increased activity at higher pH values (Figure 7C ) as expected when the deprotonation of the 5′-hydroxyl group is the likely rate-limiting step of the reaction. The temperature optimum of the 44-nucleotide ribozyme is around 10°C (Figure 7D ), different from the optimum of a TPR1 variant at 40°C ( 49 ), underlining the different characteristics of these two ribozymes. Together, the optimum reaction conditions for the 44-nucleotide long ribozyme were 200 mM Tmp, 100 mM Mg 2+ , a pH of 8.5, and a temperature of 10°C.…”

A Peptide–Nucleic Acid Replicator Origin for Life

“…The most recent advance is the 24-3 ribozyme that can copy RNA sequences having secondary structure, although this is still possible only for short sequences; that is, they cannot copy themselves. These recent ribozymes, at close to 200 nt in length, are likely too big to spontaneously arise, although other recent work [41] suggests that, in some cases, ribozymes may have been able to function as fragments working together. In summary, if a self-replicating RNA-only ribozyme polymerase does prove possible, it may be that it is too long and complex to have arisen as the IDA.…”

Nucleic acids: function and potential for abiogenesis

“…The identified TPR1/TPR1e ribozyme catalyses the formation of triphosphorylated RNA from trimetaphosphate, and a 5′-hydroxyl RNA oligonucleotide with a catalytic rate of 6·8 min −1 under optimal conditions. Originally 96 nt long, a recently derived fragmented variant can be constructed from oligonucleotides no longer than 34 nt (Akoopie & Muller, 2016), approaching the range of RNA oligomers accessible by non-enzymatic RNA polymerization (Ferris et al 1996). However, none of the current variants is capable of directly triphosphorylating nucleoside monomers and relies on attachment as part of a polynucleotide for 5′ positioning; general nucleoside substrate binding may be a challenging trait to evolve due to the tendency for RNA molecules to harness base-pairing for molecular recognition.…”

“…Indeed, in vitro selection experiments suggest that functional sequences are extremely rare (Szostak, 2003) although some very small RNAs can display catalytic function such as the aminoacylating 5 nt ribozyme (Turk et al 2011). Furthermore, larger ribozymes such as the hairpin ribozyme (Vlassov et al 2004) and a triphosphorylation ribozyme (Akoopie & Muller, 2016) can retain function and near wild-type catalytic rates when fragmented into 20–30 nt pieces, which are within the size range accessible from prebiotic chemistry and non-enzymatic replication. Thus, simple ribozymes, may be able to emerge from pools of short oligomers either directly or by non-covalent assembly into functional units and this might allow the bootstrapping of oligomer pools towards the higher compositional and functional complexity needed for self-replication.…”

“…<30 nt long), can be more easily separated into product and template strands after replication. While some simple ribozymes are able to self-assemble from RNA fragments in this size range (Akoopie & Muller, 2016; Vlassov et al 2004), this does not appear to be generally the case, in particular for more complex ribozymes. Indeed, fragmentation and non-covalent assembly of the R18-derived RPR into multiple fragments dramatically reduces activity, and therefore the covalent assembly through a ligase (or recombinase) ribozyme would be required.…”