RNA ligase ribozymes with a small catalytic core

Abstract

Catalytic RNAs, or ribozymes, catalyze diverse chemical reactions that could have sustained primordial life in the hypothetical RNA world. Many natural ribozymes and laboratory evolved ribozymes exhibit efficient catalysis mediated by elaborate catalytic cores within complex tertiary structures. However, such complex RNA structures and sequences are unlikely to have emerged by chance during the earliest phase of chemical evolution. Here, we explored simple and small ribozyme motifs capable of ligating two RNA fragments in a template-directed fashion (ligase ribozymes). One-round selection of small ligase ribozymes followed by deep sequencing revealed a ligase ribozyme motif comprising a three-nucleotide loop opposite to the ligation junction. The observed ligation was magnesium(II) dependent and appears to form a 2'-5' phosphodiester linkage. The fact that such a small RNA motif can function as a catalyst supports a scenario in which RNA or other primordial nucleic acids played a central role in chemical evolution of life.

Figure 1

Ribozyme library design and analysis. (a) Secondary structures of F1* and 4d394 ligase ribozymes with cognate substrates. Catalytic cores are shown in red. (b) Ribozyme library Lib-N7/8. Nucleotides shown in red are randomized. (c) Outline of the sequencing library construction. The ligated and unligated RNA strands are first reverse transcribed (RT) to cDNAs and then amplified by PCR. Another PCR step adds adapter sequences for analysis by MiSeq.

Figure 2

Characterization of N8-1 catalysis. (a) Predicted secondary structure of N8-1 in the presence of T7Psub by NUPACK. (b) Ligation kinetics of N8-1 with T7Psub in the presence of 10, 25, and 50 mM MgCl₂. First-order rate constants (k_obs) were estimated by calculating the initial rates of the reactions by fitting the data to linear (10 and 25 mM) or third order polynomial functions (50 mM). The error bars indicate the range of two measurements performed independently. (c) Digestion of the N8-1 ligation product by Dz8-17-N8-1 for analysis of the ligation regioselectivity. Lane 1: FAM-T7Psub as a size marker. Lane 2: N8-1 RNA (unlabeled, not visible). Lane 3: FAM-T7Psub-N8-1 ligation product produced by T4 RNA ligase, digested by Dz8-17-N8-1. Lane 4: FAM-T7Psub-N8-1 ligation product catalyzed by N8-1, digested by Dz8-17-N8-1. The lower bands marked by a star are from the loading dye used.

Figure 3

Lib-N5 selection. (a) Lib-N5 design. (b) Consensus sequence (frequency plot) of the 18 sequences that yielded enrichment of 10 or greater. The logo was generated by WebLogo. (c) Ligation junctions of N8-1, a4-20, and J4 ribozymes.

Figure 4

Sequence requirement of N8-1 at the 5′ end. (a) Nucleotides mutated (N) in N8-1. (b) Ligation yields of N8-1 mutants. The first 3 nucleotides of the mutants are listed. The yields were measured after 4 h reaction at 42 °C in the presence of 25 mM MgCl₂. The error bars represent the range of two independent measurements.