Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles.

Yoko Nomura,Yohei Yokobayashi

doi:10.1093/nar/gkz729

Abstract

Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.

Highlights

No direct molecular signatures exist, it is widely accepted that RNA preceded DNA and proteins in the hypothetical RNA world [1,2] in which RNA assumed the roles of storing and transferring genetic information as well as catalyzing essential biochemical reactions to sustain life
We found that a catalytic core as small as 18 bases can catalyze the reaction at a moderate rate, which represents the smallest catalytic core for this class of ribozymes
The applied selection pressure determines the arbitrary threshold of catalytic activity which is difficult to control

Summary

Introduction

No direct molecular signatures exist, it is widely accepted that RNA preceded DNA and proteins in the hypothetical RNA world [1,2] in which RNA assumed the roles of storing and transferring genetic information as well as catalyzing essential biochemical reactions to sustain life. One RNA ligase ribozyme (class I Bartel ligase) that was discovered through in vitro selection from a large pool of random RNA sequences can catalyze template-directed RNA ligation very efficiently (kobs > 1 s−1) [3]. This rather large (∼180 nt) and structurally complex ligase was further engineered to function as RNA polymerase ribozymes through a series of laboratory evolution experiments [4,5,6,7,8].

Methods

Results

Conclusion