In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

Abstract

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozymecatalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.ribozymes | ligation | prebiotic T he RNA world hypothesis proposes a central role for RNA as both a catalytic and an informational polymer during the emergence of life. Evidence for the RNA world includes a variety of functional RNAs, such as ribozymes and riboswitches, as well as the prebiotically plausible syntheses of nucleotides (1, 2). An important tenet of the RNA world hypothesis is that informational and functional RNA polymers must have preceded complex protein enzymes, as exemplified by the RNA-catalyzed synthesis of proteins inside the catalytic core of the ribosome (3). However, the antecedence of functional RNA raises fundamental questions regarding the formation and replication of RNA polymers during the origin of life, when protein enzymes were absent. Under nonenzymatic reaction conditions, the formation of phosphodiester bonds using biological nucleoside-5′-triphosphates (NTPs) is slower than the hydrolysis of phosphodiester linkages under identical conditions, which would severely limit the accumulation of RNA polymers (4, 5).Experimental studies have instead utilized a variety of intrinsically reactive monomers and oligomers to form both random single-stranded polymers and templated double-stranded RNA products (6-8). In particular, imidazoles have been extensively studied as the leaving group of RNA monomers called nucleoside-5′-phosphor-imidazolides (9, 10). Recent work has demonstrated that 2-aminoimidazole (...

tRNA sequences can assemble into a replicator

“…The RNA world hypothesis proposes, that early on, the catalytic function of highly defined RNA sequences was used for self-replication ( Horning and Joyce, 2016 ; Orgel, 2004 ; Turk et al, 2011 ). These ribozymes catalyze the ligation of RNA ( Doudna et al, 1991 ; Mutschler et al, 2015 ; Paul and Joyce, 2002 ; Robertson et al, 2001 ; Walton et al, 2020 ) and the addition of individual bases ( Attwater et al, 2013 ; Horning and Joyce, 2016 ). These very special sequences were engineered using in vitro evolution.…”

New Deoxyribozymes for the Native Ligation of RNA

“…In the best-case scenario, crystal structure analyses may help disclose a recognition nucleotide for the adenylated 5 -guanosine and suggest compensatory mutations to further expand the substrate scope of SC9. Additional future directions consider the evolution of bifunctional nucleic acid catalysts that may catalyze the activation and the ligation steps (for example, by using ATP as cofactor [46]), or that use alternative activated donor substrates, such as phosphor-2-aminoimidazolides that were recently explored as prebiotically plausible substrates for RNA-ligating ribozymes [47].…”

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…Most commonly, 5 0 -triphosphates are used, but 5 0 -adenylates, nicotinamides, or substituted 5 0 -phosphorimidazolides have also been employed. [269][270][271] Fundamentally, this reaction resembles the elongation step catalysed by RNA polymerases, and is thus also of great interest in the context of origin of life research and the RNA world, in which an RNA polymerase ribozyme would have been able to copy functional RNA molecules. 118,272…”