Intracellular selection of trans-cleaving hammerhead ribozymes.

Xin Huang,Zhuo Tang,Xin Cui,Meiling Sun,Qinlin Pu,Feng Du,Xianming Mo,Yan Peng,Fei Wang,Yongyun Zhao,Gangyi Chen,Juan Dong,Getong Liu

doi:10.1093/nar/gkz018

Xin Huang, Zhuo Tang + Show 11 more

Open Access

https://doi.org/10.1093/nar/gkz018

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Abstract

Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.

Highlights

Catalytic RNAs were termed ribozymes because of their similar catalytic property to protein enzymes
Based on the unique secondary structure, hammerhead ribozymes have been engineered to cleave any given RNA by an intermolecular attack, through recognizing substrate by two binding arms and cleaving adjacent to the sequence NUX↓ (N is any base and X is A, C or U) [4]. Small ribozymes, such as Hairpin or hepatitis delta virus (HDV), have trans-cleaving potential to be applied in the trans-cleavage of targeted mRNA for suppression of gene expression [5,6], HHRz is the most attractive candidate for the development of ribozyme-based therapeutic tools because of its small size and flexibility in design [7,8,9,10,11,12]
We describe a completely direct intracellular selection method to evolve HHRz variants based on trans-cleavage mode via using a toxin gene as the reporter

Summary

Introduction

Catalytic RNAs were termed ribozymes because of their similar catalytic property to protein enzymes. Based on the unique secondary structure, hammerhead ribozymes have been engineered to cleave any given RNA by an intermolecular attack (trans-cleavage), through recognizing substrate by two binding arms and cleaving adjacent to the sequence NUX↓ (N is any base and X is A, C or U) [4]. Small ribozymes, such as Hairpin or hepatitis delta virus (HDV), have trans-cleaving potential to be applied in the trans-cleavage of targeted mRNA for suppression of gene expression [5,6], HHRz is the most attractive candidate for the development of ribozyme-based therapeutic tools because of its small size and flexibility in design [7,8,9,10,11,12]. To improve the antiviral efficacy in future clinical trials, the identification of new ribozymes and RNA conjugation strategies is required [15]

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