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.     

Specificity of hammerhead ribozyme cleavage.

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

Pseudoknot Interaction-Mediated Activation of Type I Hammerhead Ribozyme: A New Class of Gene-Therapeutic Agents

Recently discovered hammerhead ribozymes that are activated through pseudoknot interactions (Watson-Crick base pairs between loops) are attractive candidates as gene-therapeutic agents because sequences of gene-therapeutic ribozymes can be designed simply based on the sequence complementarity against target RNAs. Herein, we examined if the newly found pseudoknot-type hammerhead ribozyme with type I topology is activated through the pseudoknot interactions. Substitutions of pseudoknot sequences into fully mismatched ones significantly reduced the activity of type I pseudoknot-type hammerhead ribozyme, while those with full-matched pseudoknot sequences were highly active. The results indicated that the pseudoknot interactions activated type I pseudoknot-type hammerhead ribozyme, making them suitable as gene-therapeutic agents.     

Design and specificity of hammerhead ribozymes against calretinin mRNA.

We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.     

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.     

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.     

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.     

Induction of ribozyme activity by anti-ribozyme oligonucleotides.

A new type of hammerhead ribozyme, with cleavage activity enhanced by oligonucleotides, was constructed. Stem II of the ribozyme was substituted with a non complementary loop (loop II). The modified ribozyme exhibited negligible cleavage of a target RNA; however, it was converted to an active molecule in the presence of oligonucleotides which were complementary to loop II. The oligonucleotide compensated for the disabled stem II by binding with the ribozyme. The induction of the cleavage activity was sequence-specific and the oligonucleotides containing a purine base as the 3'-dangling end were able to induce the cleavage activity of the ribozyme most efficiently. A photo-crosslinking experiment proved that a pseudo-half-knot structure was formed in the active molecule. The cleavage of two kinds of substrate RNAs with different sequences was controlled by the corresponding ribozymes activated by specific oligonucleotides.     

抗HPV11 E2核酶的计算机设计

 借助计算机软件分析 ,设计出能特异性切割HPV11型 6 4 4ntE2mRNA的核酶 (ribozyme) .遵循Symon′s锤头状核酶结构和GUX剪切位点原则 ,靶序列存在 32个这样的剪切位点 .通过计算机软件分析出核酶的最佳剪切位点 ,并对底物及核酶的二级结构进行预测及进行相应基因生物学功能和基因同源性分析 ,筛选出 2个锤头结构核酶 .针对这两位点设计的核酶分别命名为RZ2 777和RZ32 81.计算机分析显示 ,两核酶与底物切点两翼碱基形成锤头状结构 ,切点所在基因序列具有相对松弛的二级结构 ,位于该基因重要生物功能区内 ,是核酶的理想攻击区域 .通过基因库检索 ,在已知人类基因排除了与上述两核酶切点两翼碱基有基因同源性序列的可能性 .将两核酶用于体外剪切实验取得了良好的实验结果 ,认为借助计算机分析可帮助尽快从多个剪切位点选择出最适核酶     

Inosine(15.1) hammerhead ribozymes for targeting the transthyretin-30 mutation.

The most common cause of hereditary amyloidosis (HA) is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of our work was the development of a specific cleavage of TTR-30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable hammerhead ribozymes to target the TTR-30 mRNA with high specificity. The exchange of adenosine(15.1) with inosine(15.1) in the catalytic core of the hammerhead ribozyme resulted in a change of the cleavable target sequence from N(16.2)U(16.1)H(17) to N(16. 2)C(16.1)H(17) without loss in ribozymal activity (Nucleic Acids Res. 26, 2279-2285, 1998). This modification allowed a specific cleavage of the TTR-30 mutation ("gCC Gug" to "gCC Aug"). In vitro experiments with TTR-30 mRNA demonstrated that the RNase stable inosine(15.1) hammerhead ribozyme cleaved the TTR-30 mRNA with 100% specificity and with a velocity of 0.23 min(-1), whereas no cleavage occured in the wildtype mRNA of TTR. In conclusion, the development of this NCH specific hammerhead ribozyme represents a promising tool for future in vivo therapeutic application for TTR-met30 induced hereditary amyloidosis.     

Binary hammerhead ribozymes with high cleavage activity

Binary hammerhead ribozymes consisted of two oligoribonucleotides capable of assembling into hammerhead structure (without loop II) on the RNA target were engineered. Catalytic activities of such ribozymes were investigated in comparison with their full-length analog and ribozyme where two strands were jointed by non-nucleotidic linker. Binary constructs were shown to be significantly more active than the parent full-length hammerhead ribozyme.     

Small, efficient hammerhead ribozymes

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5′ and 3′ hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5′ arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix+loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5′ and the 3′ arms of a miniribozyme containing an optimized sequence for helix+loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of genelength RNA substrates may be best achieved by miniribozymes.     

Binary hammerhead ribozymes with improved catalytic activity

A new design of binary hammerhead ribozymes displaying high catalytic activity and nucleolytic stability is described. These catalytic structures consist of two partially complementary oligoribonucleotides, capable of assembling into the hammerhead-like structure without tetraloop II on binding to the RNA target. A series of these binary ribozymes targeting the translation initiation region of multiple drug resistance gene mdr1 mRNA was synthesized and assessed in terms of catalytic activity under single and multiple reaction turnover conditions. Enhanced nuclease resistance of the binary ribozymes was achieved by incorporation of 2'-modified nucleotides at selected positions, along with addition of a 3'-3'-linked thymidine cap. The new binary ribozymes exhibit higher RNA cleavage activity than their full-length analogs because of faster dissociation of cleavage products. Furthermore, an excess of one of the ribozyme strands provides the possibility to unfold structured regions of the target RNA and facilitate productive complex formation.     

An ultraviolet crosslink in the hammerhead ribozyme dependent on 2-thiocytidine or 4-thiouridine substitution.

The hammerhead domain is one of the smallest known ribozymes. Like other ribozymes it catalyzes site-specific cleavage of a phosphodiester bond. The hammerhead ribozyme has been the subject of a vast number of biochemical and structural studies aimed at determining the structure and mechanism of cleavage. Recently crystallographic analysis has produced a structure for the hammerhead. As the hammerhead is capable of undergoing cleavage within the crystal, it would appear that the crystal structure is representative of the catalytically active solution structure. However, the crystal structure conflicts with much of the biochemical data and reveals a catalytic metal ion binding site expected to be of very low affinity. Clearly, additional studies are needed to reconcile the discrepancies and provide a clear understanding of the structure and mechanism of the hammerhead ribozyme. Here we demonstrate that a unique crosslink can be induced in the hammerhead with 2-thiocytidine or 4-thiouridine substitution at different locations within the conserved core. Generation of the same crosslink with different modifications at different positions suggests that the structure trapped by the crosslink may be relevant to the catalytically active solution structure of the hammerhead ribozyme. As this crosslink appears to be incompatible with the crystal structure, this provides yet another indication that the active solution and crystal structures may differ significantly.     

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.     

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.     

Activity identification of chimeric anti-caspase-3 mRNA hammerhead ribozyme in vitro and in vivo

To study the expression activity of various vectors containing anti-caspase-3 ribozyme cassettes in vivo, and to further study the role of caspas-3 in the apoptotic pathway, we constructed anti-caspase-3 hammerhead ribozyme embedded into the human snRNA U6, and detected the activity of the ribozyme in vitro and in vivo. Meanwhile we compared it with the self-cleaving hammerhead ribozymes that we previously studied, and with the general ribozyme, cloned into RNA polymerase II expression systems. The results showed that the three ribozymes, p1.5RZ107, pRZ107 and pU6RZ107 had the correct structure, and that they could cleave cas-pase-3 mRNA exactly to produce two fragments: 143nt/553nt. p1.5RZ107 has the highest cleavage efficiency in vitro, almost 80%. However, the U6 chimeric ribozyme, pU6RZ107, has the highest cleavage activity in vivo, almost to 65%, though it has lower cleavage activity in vitro. The cleavage results demonstrated that the pU6RZ107, the U6 chimeric ribozyme, could more efficiently expre     

Binary Hammerhead Ribozymes with High Cleavage Activity

Binary hammerhead ribozymes consisted of two oligoribonucleotides capable of assembling into hammerhead structure (without loop II) on the RNA target were engineered. Catalytic activities of such ribozymes were investigated in comparison with their full‐length analog and ribozyme where two strands were jointed by non‐nucleotidic linker. Binary constructs were shown to be significantly more active than the parent full‐length hammerhead ribozyme.     

Comparative single-turnover kinetic analyses of trans-cleaving hammerhead ribozymes with naturally derived non-conserved sequence motifs

trans-Cleaving hammerhead ribozyme variants were generated with mimicked non-conserved internal loop motifs derived from five structurally diverse natural cis-cleaving ribozymes. Most modified trans-cleaving variants showed enhanced single-turnover cleavage rates relative to minimal counterparts that lack tertiary interactions between internal loop motifs I and II, and relative to controls with sequence changes in loop I. The trans-cleaving ribozyme derived from the positive strand of peach latent mosaic viroid had the highest observed cleavage rate, suggesting a structurally optimized motif that facilitates rapid formation of the ribozyme catalytic center in a trans-reaction.