Optimizing the substrate specificity of a group I intron ribozyme

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.     

Modulating the splicing activity of Tetrahymena ribozyme via RNA self-assembly

The internal guiding sequence (IGS) is normally located at the 5' end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.     

Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.     

截短绿色荧光蛋白突变体反式剪接修复核酶

Ⅰ型内含子核酶经过设计特定的信号引导序列（IGS）,可特异性地定点剪接目的基因RNA,从而在RNA水平达到修复病变基因的目的。以四膜虫材料,克隆了其26S rRNA内含子核酶基因,体外转录证实该I型内含子核酶具有完全的自我剪接的功能。为检测该核酶的反式剪接功能,构建了缺失后半段564bp基因序列的绿色荧光蛋白（GFP）的截短突变体重组质粒XYQ5/XYQ10pEGFP-C-2,并证实其失去了发射绿色荧光的活性。利用PCR和分子克隆技术,构建了以上EGFP突变体的反式剪接修复核酶ptrans-rib-CMV2,该核酶载体以克隆的26S RNA内含子为核心,选择EGFP编码区194位TG为剪接位点,以188-193位设计IGS序列,核酶3′端携带195-890bp的EGFP基因序列,连接于pRC-CMV2真核表达载体中。体外转录突变EGFP的原核表达载体XYQ5/10-pGEM和ptrans-rib-CMV₂,以混合转录产物为模板进行RT-PCR,电泳及测序证实产物中含有反式剪接修复的野生型EGFP mRNA,从而证实构建的反式剪接核酶具有体外反式剪接功能。将截短突变重组质粒XYQ5/XYQ10- pEGFP-C₂与核酶质粒ptrans-rib-CMV₂共转染Hela细胞,用荧光显微镜观察转染结果,发现有少量共转染的Hela细胞发出绿光;RT-PCR检测出野生型EGFP mRNA,证明构建的反式剪接核酶具有体内反式剪接的功能,但其反式剪接效率低。     

Ribozyme inhibition of alphavirus replication

A model system to examine the expression and antiviral activity of trans-acting ribozymes in mammalian cells has been developed and evaluated. Hairpin ribozymes were engineered to cleave a specific site, identified by a combinatorial activity-based selection method, within genomic and subgenomic RNA species of Sindbis virus. Transiently transfected cells expressed moderate levels of ribozyme (approximately 50,000 molecules/cell) with predominant nuclear localization and a short half-life (23 min). Stable cell lines expressed ribozymes at modest levels (approximately 2,000 molecules/cell). Ribozyme-mediated RNA cleavage activity was detected in cell extracts. Clonal cell lines were challenged with recombinant Sindbis virus, and viral replication was examined using plaque formation and green fluorescent protein assays. Significant inhibition of viral replication was observed in cells expressing the active antiviral ribozyme, and lower levels of inhibition in control cells expressing inactive or irrelevant ribozymes. These findings are consistent with a model in which inhibition of viral replication occurs via ribozyme cleavage of viral RNAs, suggesting that ribozymes may represent useful antiviral agents.     

Suppression of BCR-ABL mRNA by various ribozymes in HeLa cells.

Ribozymes are RNA molecules with enzymatic activity that can cleave target RNA molecules in a sequence specific manner. To date, various types of ribozyme have been constructed to cleave other RNAs and such trans-acting ribozymes include hammerhead, hairpin and HDV ribozymes. External guide sequence (EGS) can also induce the suppression of a gene-expression by taking advantage of cellular RNase P. Here we compared the activities of various functional RNA cleavers both in vitro and in vivo. The first purpose of this comparison was intended to determine the best ribozyme motif with the highest activity in cells. The second purpose is to know the correlation between the activities of ribozymes in vitro and in vivo. Our results indicated that the intrinsic cleavage activity of ribozymes is not the sole determinant that is responsible for the activity of a ribozyme in cultured cells.     

Ribozyme mediated destruction of RNA in vivo.

Previous studies have demonstrated that high ribozyme to substrate ratios are required for ribozyme inhibitory function in nuclear extracts. To obtain high intracellular levels of ribozymes, tRNA genes, known to be highly expressed in most tissues, have been modified for use as ribozyme expression cassettes. Ribozyme coding sequences were placed between the A and the B box, internal promoter sequences of a Xenopus tRNAMet gene. When injected into the nucleus of frog oocytes, the ribozyme tRNA gene (ribtDNA) produces 'hammerhead' ribozymes which cleave the 5' sequences of U7snRNA, its target substrate, with high efficiency in vitro. Oocytes were coinjected with ribtDNA, U7snRNA and control substrate RNA devoid of a cleavage sequence. It was found that the ribtRNA remained localized mainly in the nucleus, whereas the substrate and the control RNA exited rapidly into the cytoplasm. However, sufficient ribtRNA migrated into the cytoplasm to cleave, and destroy, the U7snRNA. Thus, the action of targeted 'hammerhead' ribozymes in vivo is demonstrated.     

Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.