Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

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.     

2′-O-Methyl Thiomethyl Modifications in Hammerhead Ribozymes

Abstract

The synthesis of all four phosphoramidites of 2′-O-methylthiomethyl ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

Synthesis of 6-Aza- & 6-Methyl-pyrimidine Ribonucleoside Phosphoramidites and Their Incorporation in Hammerhead Ribozymes

Abstract

The synthesis of phosphoramidites of 6-modified pyrimidine ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.     

The development of a monoclonal antibody specific for a 2(')-C-allyl modification of uridine,and its use in the localization of ribozymes in vivo

Ribozymes are catalytically active RNA molecules that cleave other RNA molecules in a sequence-specific fashion, with significant turnover. The successful design and synthesis of ribozymes with modifications to increase their stability in biological fluids, while maintaining catalytic activity, has been instrumental in moving this technology from the laboratory into clinical trials. With the entry of ribozymes into the clinical setting, the need has arisen for reagents and/or assays to detect these drugs in tissues. We have developed a monoclonal antibody to the 2(')-deoxy-2(')-C-allyl uridine modification present in our synthetic hammerhead ribozymes. The monoclonal antibody, termed CA1USR, is a murine IgG1(k), whose epitope appears to involve both the 2(')-C-allyl modification, and the uridine base. Use of CA1USR for immunohistochemical detection of ribozymes in the tissues of mice which were administered two structurally different ribozymes has demonstrated its utility as a reagent for in vivo localization of ribozymes containing the 2(')-C-allyl uridine modification.     

Influence of threose nucleoside units on the catalytic activity of a hammerhead ribozyme

TNA (alpha-L-threose nucleic acids) is potentially a natural nucleic acid, that might have acted as an evolutionary alternative of RNA. We determined the catalytic activity of hammerhead ribozymes containing a threofuranosyl-modified nucleoside at position U4 and U7, and compared these results with those obtained from HNA (hexitol nucleic acids) insertion into the same ribozyme. Our experiments showed that, although the threofuranosyl-modified ribozymes still cleave the substrate strand, cleavage activity is highly decreased. It, therefore, seems that TNA can play a functional role in the RNA world, but only to a limited extent.     

Hammerhead ribozymes to modulate telomerase activity of endometrial carcinoma cells.

Telomerase is an excellent target molecule for cancer therapy, though any effective agents have never been developed in human subjects. We designed a variety of hammerhead ribozymes against human telomerase RNA (hTR) and hTERT mRNA and studied their possibility as a tool for cancer therapy. To search promising target site of hTR, the catalytic actiuity of 3 kinds of hammerhead ribozymes was studied in cell-free system. They showed equivalent catalytic activity, but only 36-ribozyme, which was designed to cleave the template region of hTR, revealed telomerase inhibitory activity in an endometrial carcinoma cell line. Among hTERT-mRNA-targeted ribozymes, the ribozyme to cleave 13 nucleotides downstream from the 5'-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to cleave 59 nucleotides upstream from the poly(A) tail showed clear activity. Stable transfection studies confirmed that the 36-ribozyme as well as the 13-ribozyme suppressed telomerase. These observations suggest that the template region of hTR and 5'end of hTERT mRNA are promising target sites for ribozymes to reduce telomerase activity.     

The tolerance to exchanges of the Watson Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson-Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson-Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3-R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem-loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson-Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes.     

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.     

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.     

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.     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.     

The effect of universal fluorinated nucleobases on the catalytic activity of ribozymes

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2, containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The Km value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM(-1), the corresponding kcat is 2.91 x 10(-3) min(-1). A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2'-(beta-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.     

Study of a hammerhead ribozyme containing 2'-modified adenosine residues

The improved synthesis of 2'-fluoro-2'-deoxyadenosine (2'-FA) starting from adenosine is described. This compound was converted to the phosphoramidite and incorporated into a hammerhead ribozyme RNA with the use of automated RNA synthesis techniques. Ribozymes containing 2'-deoxy-adenosine (2'-dA) were prepared in a similar manner. A kinetic rate comparison of the unmodified ribozyme with two ribozymes that had every adenosine replaced with 2'FA or 2'-dA revealed a large decrease in catalytic efficiency (kcat/Km) for the modified ribozymes resulting from a drop in kcat. The kinetic analysis of a number of partially substituted 2'-FA or 2'-dA containing hammerheads revealed that the decrease in activity was not associated with any particular residue but was the result of the accumulation of modified nucleosides within the structure.     

Synthesis of site-specifically modified oligoribonucleotides for studies of the recognition of TAR RNA by HIV-1 tat protein and studies of hammerhead ribozymes.

Synthetic oligoribonucleotides having single uracil residues replaced by dU, dT, 2'-O-methylU or 5-bromodU have been prepared and used in the study of the interaction of HIV-1 tat protein with an RNA stem-loop. The preparation of phosphoramidites of 5-bromouridine and purine riboside suitable for use in solid-phase oligoribonucleotide synthesis is also described. The effect of adenine replacement by purine in a hammerhead ribozyme has also been determined.