Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?

Abstract

Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the help of high-throughput bioinformatic approaches. Understanding the biological role of the catalytic RNA motifs widely distributed in diverse genetic contexts belongs to the big challenges of future RNA research.

Keywords: RNA; catalytic; ribozyme.

Figure 1.

Ribozyme-based ON ( a) and OFF ( b) switches. The ribozyme-based device is positioned in the 5′-untranslated region (5′-UTR) of the transcript of interest. ( a) In the absence of a specific ligand, the ribozyme is inactive and the ribosome-binding site (RBS) is sequestered in a double-stranded region; translation is switched OFF. Upon ligand binding, the ribozyme is activated and cleavage can take place. As a result, the RBS is set free and translation can proceed. ( b) In the absence of a specific ligand, the ribozyme undergoes self-cleavage, thereby freeing the RBS and allowing translation to proceed. Binding of the ligand inhibits ribozyme activity, and translation is switched OFF.

Figure 2.. Selection of a DNAzyme from a random library under varying conditions.

For more detail, see the ‘DNAzymes’ section of the main text.

Figure 3.. Structure of xeno nucleic acids in comparison with DNA and RNA.

ANA, arabino nucleic acid; CeNA, cyclohexene nucleic acid; FANA, 2′-fluoroarabino nucleic acid; HNA, hexitol nucleic acid.

Figure 4.. Schematic presentation of ribozyme activities that might have played a role in the RNA world.

a) self-modification, e.g. alkylation; b) 5'-terminal modification by ribozyme-supported addition of an activated building block; c) internal modification by ribozyme-supported fragment exchange; d) ribozyme-supported 5' –triphosphorylation with trimetaphosphate; e) ribozyme-supported RNA polymerization with nucleoside-2',3'-cyclic phosphates (in 3'→5'-direction) or nucleoside-5'-triphosphates (in 5'→3'-direction) as activated building blocks.

Figure 5.. Secondary structures of recently discovered ribozymes.

The arrows denote the cleavage sites.