Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes.

The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min-1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridines. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A7, A9 and A10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the ribozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency

A root-specific condensing enzyme from Lesquerella fendleri that elongates very-long-chain saturated fatty acids

NRC publication: Ye

A root-specific condensing enzyme from Lesquerella fendleri that elongates very-long-chain saturated fatty acids

NRC publication: Ye

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs