Engineered RNase P ribozymes increase their cleavage activities and efficacies in inhibiting viral gene expression in cells by enhancing the rate of cleavage and binding of the target mRNA

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.     

Effective Inhibition of Human Immunodeficiency Virus 1 Replication by Engineered RNase P Ribozyme

Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the tat region. The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G₈₃ -> U₈₃ and G₃₄₀ -> A₃₄₀) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.     

RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.     

Developing RNase P ribozymes for gene-targeting and antiviral therapy

RNase P, a tRNA processing enzyme, contains both RNA and protein subunits. M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, recognizes its target RNA substrate mainly on the basis of its structure and cleaves a double stranded RNA helix at the 5' end that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. Accordingly, a guide sequence (GS) can be covalently attached to the M1 RNA to generate a sequence specific ribozyme, M1GS RNA. M1GS ribozyme can target any mRNA sequence of choice that is complementary to its guide sequence. Recent studies have shown that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1 and human cytomegalovirus, and the BCR-ABL oncogenic mRNA in vitro and effectively reduce the expression of these mRNAs in cultured cells. Moreover, an in vitro selection scheme has been developed to select for M1 GS ribozyme variants with more efficient catalytic activity in cleaving mRNAs. When expressed in cultured cells, these selected ribozymes also show an enhance ability to inhibit viral gene expression and growth. These recent results demonstrate the feasibility of developing the M1GS ribozyme-based technology as a promising gene targeting approach for basic research and clinical therapeutic application.     

In vitro construction of effective M1GS ribozymes targeting HCMV UL54 RNA segments

Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3′ terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site-1; and (4) around the cleavage site, the sites -1/ 1/ 2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed.Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.     

Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human cytomegalovirus (HCMV) protease (PR), a viral protein that is responsible for the processing of the viral capsid assembly protein. We showed that the constructed ribozyme cleaved the PR mRNA sequence efficiently in vitro. Moreover, a reduction of about 80% in the expression level of the protease and a reduction of about 100-fold in HCMV growth were observed in cells that expressed the ribozyme stably. In contrast, a reduction of less than 10% in the expression of viral protease and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Further examination of the antiviral effects of the ribozyme-mediated cleavage of PR mRNA indicates that (1) the proteolytic cleavage of the capsid assembly protein is inhibited significantly, and (2) the packaging of the viral genomic DNA into the CMV capsids is blocked. These observations are consistent with the notion that the protease functions to process the capsid assembly protein and is essential for viral capsid assembly. Moreover, our results indicate that the RNase P ribozyme-mediated cleavage specifically reduces the expression of the protease, but not other viral genes examined. Thus, M1GS ribozyme is highly effective in inhibiting HCMV growth by targeting the PR mRNA and may represent a novel class of general gene-targeting agents for the studies and treatment of infections caused by human viruses, including HCMV.     

Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes

Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.     

Intracellular expression of engineered RNase P ribozymes effectively blocks gene expression and replication of human cytomegalovirus

A ribozyme (M1GS RNA) constructed from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping region of two human cytomegalovirus (HCMV) mRNAs, which encode for the viral essential protease (PR) and capsid assembly proteins (AP), respectively. The results show a reduction of >80% in the expression levels of PR and AP and an inhibition of approximately 2000-fold of viral growth in cells that stably expressed the ribozyme. In comparison, <10% reduction in the expression of the targets and viral growth was found in cells that either did not express the ribozyme or produced a "disabled" ribozyme carrying mutations that abolished its catalytic activity. Examination of replication of the virus in the ribozyme-expressing cells indicates that packaging of the viral genomic DNA into capsids is blocked, and suggests that the antiviral effects are because the ribozyme specifically inhibits the AP and PR expression and, consequently, abolishes viral capsid formation and growth. Our results show that RNase P ribozymes are highly effective in blocking HCMV growth by targeting the PR and AP mRNAs and demonstrate the feasibility to use these ribozymes in gene therapy for antiviral applications.     

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a ‘disabled’ ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.     

Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection

Self-cleaving ribozymes associated with the glmS genes of many Gram-positive bacteria are activated by binding to glucosamine-6-phosphate (GlcN6P). Representatives of the glmS ribozyme class function as metabolite-sensing riboswitches whose self-cleavage activities down-regulate the expression of GlmS enzymes that synthesizes GlcN6P. As with other riboswitches, natural glmS ribozyme isolates are highly specific for their target metabolite. Other small molecules closely related to GlcN6P, such as glucose-6-phosphate, cannot activate self-cleavage. We applied in vitro selection methods in an attempt to identify variants of a Bacillus cereus glmS ribozyme that expand the range of compounds that induce self-cleavage. In addition, we sought to increase the number of variant ribozymes of this class to further examine the proposed secondary structure model. Although numerous variant ribozymes were obtained that efficiently self-cleave, none exhibited changes in target specificity. These findings are consistent with the hypothesis that GlcN6P is used by the ribozyme as a coenzyme for RNA cleavage, rather than an allosteric effector.     

HCMV UL97 mRNA序列特异性M1GS的构建及其体外切割活性研究

HCMV UL97基因编码一种蛋白激酶,该酶参与调控病毒DNA的复制和衣壳的形成,且序列异常保守,可作为抗HCMV治疗的重要靶位。基于HCMV UL97 mRNA T3位点附近的序列,设计一段与该位点互补的引导序列（Guide Sequence,GS）,并将其与大肠杆菌核酶P催化亚基（M1 RNA）的3’末端共价连接,构建了一种序列特异性的M1GS（M1-T3）。体外实验证实,所构建的M1-T3可与UL97 mRNA的T3位点特异性结合并产生有效的切割作用。进一步研究M1-T3的结构与其对底物片段靶向切割活性的关系,结果发现在M1 RNA与GS之间增加一段88核苷酸桥连序列的M1-T3（即M1-T3’）,其靶向切割活性大大增强。此外,去除M1-T3 3’末端的CCA序列,其靶向切割活性将基本丧失。上述结果表明,这段桥连序列和3’末端的CCA序列是M1-T3重要的结构元件。这不仅有助于阐明M1GS与其底物的相互作用机制,同时也为进一步评价M1-T3在体内对UL97基因表达及病毒复制的抑制活性奠定了基础。     

BCR3/ABL2核酶的设计、构建及对K562细胞增殖与集落形成的抑制作用和凋亡诱导作用

利用计算机模拟设计合成了针对 K5 62细胞致癌融合 bcr3/abl2 m RNA的锤头状核酶 .该核酶以融合点附近 UUC为识别切割三联体 ,在核酶的 3′端增加一段 T7噬菌体终止子序列 .用基因克隆结合体外转录的方法 ,肯定了核酶的体外切割活性 .进而将核酶基因克隆到 p CEP4真核细胞高效表达载体上 ,利用脂质体 Lipofectin AMINE介导的转染技术将核酶与核酶基因导入靶细胞 ,从抑制靶细胞 K5 62的增殖与集落形成及引起靶细胞凋亡等方面验证了核酶在细胞水平上对融合基因 bcr3/abl2 m RNA的特异切割作用 ,并观察到了 T7噬菌体终止子序列对核酶切割效率的增强影响 .     

GS介导RNase P对人巨细胞病毒ul54基因mRNA的切割作用

引导序列(Guide Sequences,GSs)是与mRNA靶序列互补并引导RNase P切割的小RNA片段。设计与人巨细胞病毒HCMV(Human Cytomegalovirus,HCMV)ul54基因D片段mRNA序列互补的GS,将其共价结合到大肠杆菌来源RNase P催化核心M1 RNA,构建成T7-M1GS核酶。通过对ul54基因D片段转录产物体外切割实验和将T7-M1GS构建在含有U6启动子的逆转录病毒载体,与构建在真核载体pEGFP-N1的ul54基因D片段共转染人宫颈癌细胞系HeLa的体内切割实验,证实该核酶具备对ul54基因D片段mRNA的特异切割能力,为利用核酶治疗HCMV感染提供实验基础。     

Engineered external guide sequences effectively block viral gene expression and replication in cultured cells

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. We have previously used an in vitro selection procedure to generate EGS variants that efficiently direct human RNase P to cleave a target mRNA in vitro. In this study, a variant was used to target the mRNA encoding the protease of human cytomegalovirus (HCMV), which is essential for viral capsid formation and replication. The EGS variant was about 35-fold more active in inducing human RNase P to cleave the mRNA in vitro than the EGS derived from a natural tRNA. Moreover, a reduction of 95% in the expression of the protease and a reduction of 4,000-fold in viral growth were observed in HCMV-infected cells that expressed the EGS variant, whereas a reduction of 80% in the protease expression and an inhibition of 150-fold in viral growth were detected in cells that expressed the EGS derived from a natural tRNA sequence. No significant reduction in viral protease expression or viral growth was observed in cells that either did not express an EGS or produced a "disabled" EGS, which carried nucleotide mutations that precluded RNase P recognition. Our results provide direct evidence that engineered EGS variant is highly effective in blocking HCMV expression and growth by targeting the viral protease. Furthermore, these results demonstrate the utility of engineered EGS RNAs in gene targeting applications, including the inhibition of HCMV infection by blocking the expression of virus-encoded essential proteins.     

UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.     

Development of ribozymes that target stathmin, a major regulator of the mitotic spindle

Stathmin is a major cytosolic phosphoprotein that plays an important role in the control of cellular proliferation by regulating the dynamics of the microtubules that make up the mitotic spindle. Because stathmin is expressed at high levels in all human cancers, it is an attractive molecular target for anticancer interventions. We had shown previously that antisense stathmin inhibition results in marked abrogation of the transformed phenotype of leukemic cells in vitro and in vivo. Unlike the antisense approach, ribozymes can catalytically cleave several molecules of target RNA. This may provide a more efficient strategy for downregulating genes, such as stathmin, that are expressed at very high levels in cancer cells. We designed several antistathmin hammerhead ribozymes and tested their cleavage activity against short synthetic stathmin RNA substrates. In vitro cleavage studies demonstrated site-specific cleavage of stathmin RNA that was dependent on ribozyme concentration and duration of exposure to ribozyme. The most active antistathmin ribozyme was capable of cleaving >90% stathmin RNA in a catalytic manner, cleaving multiple substrate molecules per ribozyme molecule. We also demonstrated that the designed antistathmin ribozymes are capable of selectively cleaving native stathmin RNA in a mixture of total RNA isolated from leukemic cells. These antistathmin ribozymes may provide a novel and effective form of gene therapy that may be applicable to a wide variety of human cancers.     

Ribozymes in the age of molecular therapeutics

Ribozymes are RNA molecules capable of sequence-specific cleavage of other RNA molecules. Since the discovery of the first group I intron ribozyme in 1982, new classes of ribozymes, each with their own unique reaction, target site specifications, and potential applications, have been identified. These include hammerhead, hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme engineering has enabled the in vitro selection of synthetic ribozymes with unique properties. This, along with advances in ribozyme delivery methods and expression systems, has led to an explosion in the potential therapeutic applications of ribozymes, whether for anti-cancer or anti-viral therapy, or for gene repair.