Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.     

Generation and application of asymmetric hammerhead ribozymes.

Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5' end the catalytic domain and at its 3' end a relatively long antisense flank that is complementary to the target RNA. Asymmetric hammerhead ribozymes can be constructed via polymerase chain reaction amplification, and rules are provided on how to select the DNA oligonucleotides required for this reaction. In addition to details on construction, we describe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to minimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determination of the cleavage rate constant. Testing for efficient cleavage is also a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for target suppression in living cells.     

A helical twist-induced conformational switch activates cleavage in the hammerhead ribozyme

We have captured the structure of a pre-catalytic conformational intermediate of the hammerhead ribozyme using a phosphodiester tether formed between I and Stem II. This phosphodiester tether appears to mimic interactions in the wild-type hammerhead RNA that enable switching between nuclease and ligase activities, both of which are required in the replicative cycles of the satellite RNA viruses from which the hammerhead ribozyme is derived. The structure of this conformational intermediate reveals how the attacking nucleophile is positioned prior to cleavage, and demonstrates how restricting the ability of Stem I to rotate about its helical axis, via interactions with Stem II, can inhibit cleavage. Analogous covalent crosslinking experiments have demonstrated that imposing such restrictions on interhelical movement can change the hammerhead ribozyme from a nuclease to a ligase. Taken together, these results permit us to suggest that switching between ligase and nuclease activity is determined by the helical orientation of Stem I relative to Stem II.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based “mix and measure” hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.     

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.     

Design, characterization and testing of tRNA3Lys-based hammerhead ribozymes

A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.     

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.     

A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.     

Structure and function of the hairpin ribozyme

The hairpin ribozyme belongs to the family of small catalytic RNAs that cleave RNA substrates in a reversible reaction that generates 2',3'-cyclic phosphate and 5'-hydroxyl termini. The hairpin catalytic motif was discovered in the negative strand of the tobacco ringspot virus satellite RNA, where hairpin ribozyme-mediated self-cleavage and ligation reactions participate in processing RNA replication intermediates. The self-cleaving hairpin, hammerhead, hepatitis delta and Neurospora VS RNAs each adopt unique structures and exploit distinct kinetic and catalytic mechanisms despite catalyzing the same chemical reactions. Mechanistic studies of hairpin ribozyme reactions provided early evidence that, like protein enzymes, RNA enzymes are able to exploit a variety of catalytic strategies. In contrast to the hammerhead and Tetrahymena ribozyme reactions, hairpin-mediated cleavage and ligation proceed through a catalytic mechanism that does not require direct coordination of metal cations to phosphate or water oxygens. The hairpin ribozyme is a better ligase than it is a nuclease while the hammerhead reaction favors cleavage over ligation of bound products by nearly 200-fold. Recent structure-function studies have begun to yield insights into the molecular bases of these unique features of the hairpin ribozyme.     

Efficient ligation of the Schistosoma hammerhead ribozyme

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.     

Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding

Mg2+ is important for the RNase activity of the hammerhead ribozyme. To investigate the binding properties of Mg2+ to the hammerhead ribozyme, cleavage rates and CD spectra for substrates containing inosine or guanosine at the cleavage site were measured. The 2-amino group of this guanosine interfered with the rate of the cleavage reaction and did not affect the amount of Mg2+ bound to the hammerhead RNA. The kinetics and CD spectra for chemically synthesized oligoribonucleotides with a Sp or Rp phosphorothioate diester bond at the cleavage site indicated that 1 mol of Mg2+ binds to the pro-R oxygen of phosphate. The binding constant for Mg2+ was about 10(4) M-1, which represents outer-sphere complexation. The hammerhead ribozyme catalyzes the cleavage reaction via an in-line pathway. This mechanism has been proved for RNA cleavage by RNase A by using a modified oligonucleotide that has an Sp phosphorothionate bond at the cleavage site. From these results, we present the reaction pathway and a model for Mg2+ binding to the hammerhead ribozyme.     

A pseudoknot ribozyme structure is active in vivo and required for hepatitis delta virus RNA replication.

The ribozymes of hepatitis delta virus (HDV) have so far been studied primarily in vitro. Several structural models for HDV ribozymes based on truncated HDV RNA fragments, which are different from the hammerhead or the hairpin/paperclip ribozyme model proposed for plant viroid or virusoid RNAs, have been proposed. Whether these structures actually exist in vivo and whether ribozymes actually function in the HDV replication cycle have not been demonstrated. We have now developed an in vivo ribozyme self-cleavage assay capable of detecting self-cleavage of dimer or trimer HDV RNA in vivo. By site-directed mutagenesis and compensatory mutations to disrupt and restore potential base pairing in the ribozyme domain of the full-length HDV RNA according to the various structural models, a close correlation between the detected in vivo and the predicted in vitro ribozyme activities of various mutant RNAs was demonstrated. These results suggest that the proposed in vitro ribozyme structure likely exists and functions during the HDV replication cycle in vivo. Furthermore, the pseudoknot model most likely represents the structure responsible for the ribozyme activity in vivo. All of the mutants that had lost the ribozyme activity could not replicate, indicating that the ribozyme activities are indeed required for HDV RNA replication. However, some of the compensatory mutants which have restored both the cleavage and ligation activities could not replicate, suggesting that the ribozyme domains are also involved in other unidentified functions or in the formation of an alternative structure that is required for HDV RNA replication. This study thus established that the ribozyme has important biological functions in the HDV life cycle.     

Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs.     

抗HPV11／E2基因Ribozyme对靶RNA的体外剪切研究

为了寻找HPV11型引起的生殖系统感染的治疗途径和探讨HPV的致病机理,本实验以HPV11病毒质粒为模板,扩增出HPVll型E2区644bp片段,采用pGEM-T-Easy Vector为载体,构建pTV-644克隆载体,经筛选得到克隆株,提取质粒测序鉴定。采用上海生化所陈农安教授编制的锤头状Ribozyme设计软件进行计算机分析,选择Ribozyme对靶基因的最佳剪切位点,及进行基因同源性分析和生物学功能分析,选择出针对HPVllE2靶基因的RZ2777,在最适条件下进行体外剪切反应,发现人工合成和体外转录得到的Ribozyme分子均能在相应位点准确切割靶RNA分子,选择合适的反应条件切割效率达到60％以上,Km和Kcat值分别为0.63μmol/L、0.12μmol/L,RibozymeL两端的5′-cis-ribozyme和3′-cis-ribozyme自我剪切释放并未影响切割活性,但靶RNA侧翼序列影响了Ribozyme的剪切活性。实验研究表明,Ribozyme可能成为治疗HPVll型引起的尖锐湿疣的有效手段,并有望在分子水平上开辟出基因治疗HPVll病毒感染的另一新天地。     

丙型肝炎病毒特异性核酶对HepG2.9706细胞HCV5′NCR基因表达的抑制作用

 为探讨锤头型核酶抗丙型肝炎病毒的作用 ,根据HCV 5′NCR及翻译起始区的二级结构 ,设计及合成了 1个锤头型核酶RzA .将其插入pCI neo表达载体的多克隆位点 ,构建了表达RzA的质粒pHCV RzA .采用转基因细胞模型HepG2 970 6细胞 ,评价了pHCV RzA质粒对HCV 5′NCR调控荧光素酶表达的抑制活性 .结果表明 ,在HepG2 970 6细胞中 ,pHCV RzA以序列特异性、剂量依赖性方式抑制荧光素酶基因的表达 ,抑制率达 80 % ,提示核酶有可能作为HCV感染基因治疗的一种有效途径 .     

The hammerhead ribozyme

The hammerhead ribozyme is an intriguing RNA molecule with the ability to serve as a catalyst to cleave sequence-specifically RNA molecules in an intermolecular reaction. Preferentially Mg(2+) is required for optimal activity by inducing the catalytically competent conformation and by possibly acting as an acid-base catalyst. Even though the three-dimensional structure has been elucidated details of the structure-function relationship and of the mechanism remain unanswered. The hammerhead ribozyme has stimulated the concept of the sequence-specific cleavage of mRNAs intracellularly and thus to inhibit gene expression by preventing translation. This represents an area of considerable interest as it has the potential for the development of drugs.     

HIV-1 TAR as anchoring site for optimized catalytic RNAs

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.