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.     

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.     

In vitro evolution of the hammerhead ribozyme to a purine-specific ribozyme using mutagenic PCR with two nucleotide analogues

The conventional hammerhead ribozyme cleaves RNA 3' to nucleotide triplets with the general formula NUH, where N is any nucleotide, U is uridine and H is any nucleotide except guanosine. In order to isolate hammerhead ribozyme sequences capable of cleaving 3' to the GUG triplet, we performed a mutagenic selection protocol starting with the conventional sequence of an NUH-cleaving ribozyme. The 22 nucleotides in the core and the stem-loop II region were subjected to mutagenic PCR using the two nucleotide analogues 6-(2-deoxy-beta-d-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C)][1, 2] oxazin-7-one and of 8-oxo-2'-deoxyguanosine. After five repetitions of the selection cycle, several clones showed cleavage activity. One sequence, having one deletion, showed at least a 90 times higher in trans cleavage rate than the starting ribozyme. It cleaved 3' to GUG and GUA. The sequence of this ribozyme is essentially identical with that obtained previously by selection for AUG cleavage starting with a randomised core and stem-loop II region. This identical result of two independent selection procedures supports the notion that sequences for NUR cleavage, where R is a purine nucleotide, are not compatible with the classical hammerhead structure, and that the sequence space for this cleavage specificity is very limited. The cleavage of NUR triplets is not restricted to the sequence of the substrate that was used for selection but is sequence-independent for in trans cleavage, although the sequence context influences the value for the cleavage rate somewhat. Analysis of cleavage activities indicates the importance of A at position L2.5 in loop II.     

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.     

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.     

Sequence requirements of the hammerhead RNA self-cleavage reaction.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.     

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.     

The 2 A structure of helix 6 of the human signal recognition particle RNA

BACKGROUND: The mammalian signal recognition particle (SRP) is an essential cytoplasmic ribonucleoprotein complex involved in targeting signal-peptide-containing proteins to the endoplasmic reticulum. Assembly of the SRP requires protein SRP19 to bind first to helix 6 of the SRP RNA before the signal-peptide-recognizing protein, SRP54, can bind to helix 8 of the RNA. Helix 6 is closed by a GGAG tetraloop, which has been shown to form part of the SRP19-binding site. RESULTS: The high-resolution (2.0 A) structure of a fragment of human SRP RNA comprising 29 nucleotides of helix 6 has been determined using the multiple anomalous dispersion (MAD) method and bromine-labelled RNA. In the crystal the molecule forms 28-mer duplexes rather than the native monomeric hairpin structure, although two chemically equivalent 11 base pair stretches of the duplex represent the presumed native structure. The duplex has highly distorted A-RNA geometry caused by the occurrence of several non-Watson-Crick base pairs. These include a 5'-GGAG-3'/3'-GAGG-5' purine bulge (which replaces the tetraloop) and a 5'-AC-3'/3'-CA-5' tandem mismatch that, depending on the protonation state of the adenine bases, adopts a different conformation in the two native-like parts of the structure. The structure also shows the 2'3'-cyclic phosphate reaction product of the hammerhead ribozyme cleavage reaction. CONCLUSIONS: The 29-mer RNA is the first RNA structure of the human SRP and provides some insight into the binding mode of SRP19. The observed strong irregularities of the RNA helix make the major groove wide enough and flat enough to possibly accommodate an alpha helix of SRP19. The variety of non-canonical base pairs observed enlarges the limited repertoire of irregular RNA folds known to date and the observed conformation of the 2'3'-cyclic phosphate containing Ade29 is consistent with the current understanding of the hammerhead ribozyme reaction mechanism.     

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.     

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes.

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.     

Characterization of deoxy- and ribo-containing oligonucleotide substrates in the hammerhead self-cleavage reaction

Deoxynucleotides were introduced into a substrate fragment of the hammerhead RNA self-cleaving domain. A substrate lacking the 2' hydroxyl adjacent to the cleavage site showed no detectable cleavage under a variety of reaction conditions. Competition experiments indicate that this fragment binds to the ribozyme with an affinity similar to the all RNA fragment, suggesting that the attacking 2' hydroxyl does not substantially contribute to the binding of substrate to ribozyme. Similar competition experiments with the all DNA substrate indicate a much lower affinity for the ribozyme perhaps due to the lack of other 2' hydroxyls. A substrate containing all deoxy residues except for a ribonucleotide at the cleavage site was also shown to be active.     

特异切割苹果锈果类病毒的核酶基因的克隆和转录物的体外活性测定

根据锤头型核酶的作用模式 ,设计、合成和克隆了特异切割苹果锈果类病毒ASSVd正链 (194-196位点 )或负链 (89- 91位点 )RNA的 2个短臂锤头型核酶基因 :42nt的RzASSVd(+)和 40nt的RzASSVd(- )。经转录获得核酶转录物和³²P标记的ASSVd正、负链转录物。将核酶与ASSVd混合 ,50℃或 37℃保温 3～ 4h ,进行 8%PAGE(含8mol L尿素 )和放射自显影分析。体外切割检测表明 :2个核酶均具有特异切割活性 ,其中RzASSVd(- )对ASSVd负链的切割活性较高 ,对ASSVd正链不起作用。RzASSVd(+)对ASSVd正链的切割活性较弱 ,对ASSVd负链亦不起作用。在此基础上 ,构建得到双价核酶基因pGEMRzASSVd(± )。     

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.