Enzymatic RNA Production from NTPs Synthesized from Nucleosides and Trimetaphosphate**

Abstract

A mechanism of nucleoside triphosphorylation would have been critical in an evolving “RNA world” to provide high‐energy substrates for reactions such as RNA polymerization. However, synthetic approaches to produce ribonucleoside triphosphates (rNTPs) have suffered from conditions such as high temperatures or high pH that lead to increased RNA degradation, as well as substrate production that cannot sustain replication. Previous reports have demonstrated that cyclic trimetaphosphate (cTmp) can react with nucleosides to form rNTPs under prebiotically‐relevant conditions, but their reaction rates were unknown and the influence of reaction conditions not well‐characterized. Here we established a sensitive assay that allowed for the determination of second‐order rate constants for all four rNTPs, ranging from 1.7×10−6 to 6.5×10−6 M−1 s−1. The ATP reaction shows a linear dependence on pH and Mg2+, and an enthalpy of activation of 88±4 kJ/mol. At millimolar nucleoside and cTmp concentrations, the rNTP production rate is sufficient to facilitate RNA synthesis by both T7 RNA polymerase and a polymerase ribozyme. We suggest that the optimized reaction of cTmp with nucleosides may provide a viable connection between prebiotic nucleotide synthesis and RNA replication.

Prebiotic Chemistry: The Role of Trimetaphosphate in Prebiotic Chemical Evolution

“…F. Chizzolini et al established that P 3 m is suitable for use in mild prebiotics. Under appropriate conditions, it interacted with nucleosides to generate NTP, hence boosting RNA synthesis by T7 RNA polymerase and polymerase ribozymes ( Chizzolini et al, 2021 ).…”

Non‐canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure

“…Known drawbacks of these syntheses are relatively low yields and side reactions with chemically demanding substrates [20][21][22], such as alkenefunctionalised nucleosides like showdomycin [23]. Cyclic trimetaphosphate has been used to synthesise the canonical NTPs under mild aqueous conditions, albeit at variable yields [24]. Reactive moieties of many interesting nucleobases, such as the aliphatic alcohol group in coformycin [25], might require multiple protection/deprotection steps [21,22,26].…”

Concurrent Prebiotic Formation of Nucleoside‐Amidophosphates and Nucleoside‐Triphosphates Potentiates Transition from Abiotic to Biotic Polymerization

“…[1e,4] Central to emergence of both systems is the prebiotic activation of the nucleotides to form longer polymers,both by oligomerization and ligation. Though extant biochemistry uses NTPs, [1a] and there have been attempts to synthesize NTP-analogs under plausible prebiotic conditions, [5] the difficulty in generating NTPs and the fact that they are fairly unreactive for nonenzymatic polymerization [6] has led to the reliance on other activation processes and reactive species such as phosphorimidazolide derivatives. [7] However,i nascenario where the transition from prebiotic chemistry to biotic chemistry via (proto)enzymes capable of handling NTPs is supposed to happen, [8] the question remains as to how NTPs or oligo-TP substrates can be accessed prebiotically in an efficient manner.…”