Assembly of a Ribozyme Ligase from Short Oligomers by Nonenzymatic Ligation

Abstract

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

Figure 1

Model for the transition from nonenzymatic ligation to ribozyme mediated ligation. (a) Secondary structure of the ribozyme ligase used in all experiments in this paper. Black letters are ribonucleotides; red letters represent 3′-amino-2′,3′-dideoxyribonucleotides (T is used instead of U as a 3′-amino dideoxyribonucleotide in this study for synthetic convenience). The corresponding set of five ligator oligonucleotides is shown inside the box. The purple sequence is the 5′-triphosphate substrate of the ribozyme ligase. (b) Schematic representation of the experimental design. Potentially self-replicating oligonucleotides shown in blue take part in multistep ligation reactions templated by the green splint oligonucleotides to generate the full length ligase sequence. Following dilution, the splint oligonucleotides dissociate in the 48 °C ribozyme reaction mixture, allowing folding of the ligase ribozyme. The functional ligase then ligates itself to the 5′-triphosphate oligonucleotide substrate.

Figure 2

Efficient assembly but strong inhibition of a ribozyme ligase by 10-nt RNA splints. (a) Diagram of nonenzymatic ligation of five ligator oligonucleotides directed by four 10-nt RNA splint oligonucleotides (5′-AAGUGAUAAC-3′, 5′-CUUACGUAAC-3′, 5′-CAAAGUGUUA-3′, and 5′-UCAACCCAUC-3′). (b) (Left) PAGE analysis of the assembly of the ribozyme ligase by nonenzymatic ligation. Reactions contained 20 μM of ligator 1, 25 μM of ligators 2 to 5, 25 μM of splints 1 to 4, 100 mM HEPES, pH 8.0, 100 mM NaCl, 50 mM MgCl₂, and 100 mM HEI (1-(2-Hydroxyethyl)imidazole). The ligation reaction was carried out on ice. (Right) PAGE analysis showing the lack of activity of the product ligase. The nonenzymatic ligation product was diluted 10-fold into the ribozyme reaction buffer: 50 mM bis-tris propane, pH 8.5, 25 mM MgCl₂, and 3 μM triphosphate substrate. The ligase reaction was performed at 48 °C. No ligase activity was detected. Gels depicted are representative of triplicate experiments.

Figure 3

Efficient assembly of active ribozyme ligase by 10-nt DNA splints. (a) Diagram of nonenzymatic ligation of five ligator oligonucleotides directed by four 10-nt DNA splint oligonucleotides (5′-d(AAGTGATAAC)-3′, 5′-d(CTTACGTAAC)-3′, 5′-d(CAAAGTGTTA)-3′, and 5′-d(TCAACCCATC-3′)). (b) (Left) PAGE analysis of the assembly of the ribozyme ligase by nonenzymatic ligation. (Right) PAGE analysis showing the activity of the ribozyme ligase. The conditions for both reactions, including oligonucleotide concentrations, are as in Figure 2b, except for the use of DNA splints 5 to 8. Gels depicted are representative of triplicate experiments.

Figure 4

Efficient assembly of active ribozyme ligase by 10-nt and 9-nt RNA splints with G:U wobble pairing. (a) Diagram of nonenzymatic ligation of five ligator oligonucleotides directed by three 10-mer and one 9-nt RNA splint oligonucleotides with six C to U changes (5′-AAGUGAUAAU-3′, 5′-CUUAUGUAAU-3′, 5′-UAAAGUGUUA-3′ and 5′-CAACCUAUU-3′). (b) (Left) PAGE analysis of the assembly of active ribozyme ligase by nonenzymatic ligation. (Right) PAGE analysis showing the activity of the ribozyme ligase. The conditions for both reactions, including oligonucleotide concentrations, are as in Figure 2b, except for the use of splints 9 to 12 as shown. Gels depicted are representative of triplicate experiments.

Figure 5

Efficient assembly of active ribozyme ligase by 10-nt and 8-nt RNA splints with I:C base pairing. (a) Diagram of nonenzymatic ligation of five ligator oligonucleotides directed by three 10-mer and one 8-nt RNA splint oligonucleotides with five G to I changes (5′-AAIUIAUAAC-3′, 5′-CUUAC IUAAC-3′, 5′-CAAAIUIUUA-3′, and 5′-CAACCCAU-3′). (b) (Left) PAGE analysis of the assembly of the ribozyme ligase by nonenzymatic ligation. (Right) PAGE analysis showing the activity of the ribozyme ligase. The conditions for both reactions, including oligonucleotide concentrations, are as in Figure 2b, except for the use of the splints 13 to 16 as illustrated above. Gels depicted are representative of triplicate experiments.