Building and engineering a ribozyme with tRNA synthetase activity

Abstract

Since the discovery of the first catalytic RNA in nature about 30 years ago, the study of ribozymes (ribonucleic acid enzymes) has been thriving. Numerous ribozymes have been identified, including those that play critical roles in the biological reactions essential to life such as RNA splicing, protein synthesis and gene expression. In recent years, fewer and fewer new ribozymes have been identified from nature since research interest in this field is gradually shifting toward creation of artificial ribozymes with novel catalytic functions. Generating novel ribozymes helps biochemists better comprehend a primordial RNA world where RNAs instead of proteins are dominant catalysts, and results in the production of useful biotechnology tools. In this collection of work, we will go through the process of building a ribozyme with aminoacyl-tRNA synthetase (aaRS) activity and its application as a tool enzyme in E. coli system for the site-specific incorporation of unnatural amino acids. We will also detail the engineering work on the constructed ribozyme with the attempt of improving its tRNA specificity, using the technique of in vitro selection. Although the catalytic efficiencies of engineered ribozymes are still very modest compared with protein enzymes, this work depicts a promising future of applying the ribozyme-based aaRS in production of novel peptides and proteins.