A Potential Role for Aminoacylation in Primordial RNA Copying Chemistry

Abstract

Aminoacylated tRNAs are the substrates for ribosomal protein synthesis in all branches of life, implying an ancient origin for aminoacylation chemistry. In the 1970s, Orgel and colleagues reported potentially prebiotic routes to aminoacylated nucleotides and their RNA-templated condensation to form amino acid-bridged dinucleotides. However, it is unclear whether such reactions would have aided or impeded nonenzymatic RNA replication. Determining whether aminoacylated RNAs could have been advantageous in evolution prior to the emergence of protein synthesis remains a key challenge. We therefore tested the ability of aminoacylated RNA to participate in both templated primer extension and ligation reactions. We find that at low magnesium concentrations that favor fatty acid-based protocells, these reactions proceed orders of magnitude more rapidly than when initiated from the cis-diol of unmodified RNA. We further demonstrate that amino acid-bridged RNAs can act as templates in a subsequent round of copying. Our results suggest that aminoacylation facilitated non-enzymatic RNA replication, thus outlining a potentially primordial functional link between aminoacylation chemistry and RNA replication.

Potentially prebiotic synthesis of aminoacyl-RNA via a bridging phosphoramidate-ester intermediate

“…Whilst EDC is prebiotically implausible we use it here as others have done previously as a model activating agent allowing formation of an acyl imidazolide 12 ). This peak also disappeared on treatment with alkali, consistent with the presence of an ester linkage in the product 15 .…”

“…The phosphoramidate bond can then be cleaved under mild acid conditions generating aminoacyl-RNA (5, right).4767.7, found: 4768.0 [M+H]). This peak also disappeared on treatment with alkali, consistent with the presence of an ester linkage in the product15 .Encouraged by these findings, we further synthesized two different RNA-amino acid phosphoramidates (2-L-Ala, 3'AGCGAp-L-Ala-OH and 2-D-Ala, 3'AGCGAp-D-Ala-OH) to explore their comparable reactivity and the stereoselectivity of ester formation. Based on 31 P-NMR spectra (Figure, the yields of formation of 2-L-Ala and 2-D-Ala from their parent 5'phosphoryl-RNAs were similar, suggesting that there is no stereoselectivity in the conventional synthesis of phosphoramidates of single stranded RNA.…”

Nonenzymatic assembly of active chimeric ribozymes from aminoacylated RNA oligonucleotides

“…Having previously demonstrated the enhanced template-directed ligation of aminoacylated RNAs ( 5 ), we asked whether this strategy could be employed to assemble catalytically active ribozymes containing multiple interspersed amino acid bridges. To explore the functionality of these chimeric ribozymes, we first targeted the assembly of a 37-nucleotide (nt)–long hammerhead ribozyme from three oligonucleotides via templated ligation.…”

“…However, to our knowledge, chimeric, amino acid–bridged ribozymes created with the building blocks of both proteins and RNA have not yet been reported. In our previous work, short aminoacylated RNAs were shown to form amino acid bridges in nonenzymatic ligation reactions with imidazole-activated oligonucleotides at rates much higher than observed with unmodified RNA ( 5 ). Here, we extend that approach to show that multiple ligation reactions can generate chimeric amino acid–bridged RNAs long enough to constitute catalytic RNAs.…”

“…For example, nonenzymatic aminoacylation of the 2′,3′-diol of RNA oligonucleotides with imidazole-activated amino acids affords low levels of aminoacylation ( 1 , 2 ), primarily due to the pronounced hydrolytic instability of protonated aminoacyl esters (half-life of ca. 50 to 180 min at 22 °C, pH 8, depending on the amino acid residue) ( 3 – 5 ). Alternatively, interstrand transfer of an amino acid from a 5′-phosphate mixed anhydride to the 2′,3′-diol generates aminoacylated oligonucleotides but depends on the efficient synthesis of phospho-carboxy anhydrides, which are prone to hydrolysis and CO 2 -mediated degradation ( 6 , 7 ).…”