Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

To improve catalytic activity of ribozyme on its substrate,the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted altematively regularly and the plasmid of pGEM-MDRI/MRPI used to transcribe the M DRI/MRPI(196/210) substrate containing double target sites was also constructed by DNA recombination.Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct.The cleavage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system.The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively,and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively.The multiribozyme expression system of the [Coat'A196Rz/Coat'B210Rz]5 is more significantly superior to that of the [Coat'A 196Rz/Coat'B210Rz]1 in cleavage of RNA substrate.The fractions cleaved by [Coat'A196Rz/Coat'B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference.The studies of Mg2+ on cleavage efficiency indicate that cleavage reaction is dependent on Mg2+ ions concentration.The plot of Ig(kobs) vs.Igc(Mg2+) displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg2..It suggests that Mg2+ ions play a crucial role in multi-ribozyme cleavage on the substrate.     

Relationship between catalytic activity and secondary structure of a hammerhead ribozyme: A study using thermodynamic parameters for RNA structure prediction

Abstract

The stabilization energy for the secondary structures of wild-type hammerhead and mutant ribozymes has been calculated at different salt conditions and temperatures by using the thermodynamic parameters for RNA structure prediction. The most stable structure at each condition has been searched and the obtained secondary structure is compared with the structure suggested phylogenetically or experimentally. The results indicate that the hammerhead-type secondary structure of the ribozyme and its reactivity correlate with each other. The multibranched loop containing the self-cleavage site of the ribozyme particularly should be a key structure in the hammerhead ribozyme reaction. The predicted secondary structures also suggest that the reactivity of the hammerhead ribozyme should be very much lower at 10°C than that at 37°C.     

Synthetic mammalian riboswitches based on guanine aptazyme

Allosteric hammerhead ribozymes (aptazymes) that are activated by guanine were used to control mammalian gene expression in cis and in trans. Coexpression of the two mechanistically distinct riboswitches resulted in an improved dynamic range of gene expression.     

Rationally designed allosteric variants of hammerhead ribozymes responsive to the HIV-1 Tat protein

Hammerhead ribozymes that are subject to allosteric control by small molecule and oligonucleotide effectors have been reported recently. Rational design has been an effective strategy for the creation of these ribozymes, which incorporate structurally interdependent hammerhead motifs and effector-binding sequences. In this paper we report the rational design of the first protein-responsive allosteric ribozymes that are regulated by the HIV-1 Tat. The TAR-Tat interaction of HIV-1 has the interesting feature that both Tat and arginine are able to bind to and bring about comparable conformational changes in the TAR loop. Here we describe the construction of two classes of TAR-modified hammerhead ribozymes and their response to Tat protein and to its derivatives. Instances of both allosteric activation and inhibition were found. Interestingly, the activation response was stimulated by both Tat and argininamide while the inhibitory response was stimulated by Tat and by its derivative peptide, ADP1, but not by argininamide. Overall, the extent of allosteric response in our ribozymes was modest relative to those reported for ribozymes with small molecule effectors. Future work utilizing combinatorial approaches along with elements of rational design should reveal the means by which highly efficient, protein-mediated allostery of ribozymes may be achieved.     

RNA catalysis in model protocell vesicles

We are engaged in a long-term effort to synthesize chemical systems capable of Darwinian evolution, based on the encapsulation of self-replicating nucleic acids in self-replicating membrane vesicles. Here, we address the issue of the compatibility of these two replicating systems. Fatty acids form vesicles that are able to grow and divide, but vesicles composed solely of fatty acids are incompatible with the folding and activity of most ribozymes, because low concentrations of divalent cations (e.g., Mg(2+)) cause fatty acids to precipitate. Furthermore, vesicles that grow and divide must be permeable to the cations and substrates required for internal metabolism. We used a mixture of myristoleic acid and its glycerol monoester to construct vesicles that were Mg(2+)-tolerant and found that Mg(2+) cations can permeate the membrane and equilibrate within a few minutes. In vesicles encapsulating a hammerhead ribozyme, the addition of external Mg(2+) led to the activation and self-cleavage of the ribozyme molecules. Vesicles composed of these amphiphiles grew spontaneously through osmotically driven competition between vesicles, and further modification of the membrane composition allowed growth following mixed micelle addition. Our results show that membranes made from simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg(2+) and mononucleotides without specific macromolecular transporters. This combination of stability and dynamics is critical for building model protocells in the laboratory and may have been important for early cellular evolution.     

In vitro selection of self-cleaving DNAs

Background: Ribozymes catalyze an important set of chemical transformations in metabolism, and ‘engineered’ ribozymes have been made that catalyze a variety of additional reactions. The possibility that catalytic DNAs or ‘deoxyribozymes’ can be made has only recently been addressed. Specifically, it is unclear whether the absence of the 2′ hydroxyl renders DNA incapable of exhibiting efficient enzyme-like activity, making it impossible to discover natural or create artificial DNA biocatalysts.Results: We report the isolation by in vitro selection of two distinct classes of self-cleaving DNAs from a pool of random-sequence oligonucleotides. Individual catalysts from ‘class I’ require both Cu²⁺ and ascorbate to mediate oxidative self-cleavage. Individual catalysts from class II use Cu²⁺ as the sole cofactor. Further optimization of a class II individual by in vitro selection yielded new catalytic DNAs that facilitate Cu²⁺-dependent self-cleavage with rate enhancements exceeding 1000 000-fold relative to the uncatalyzed rate of DNA cleavage.Conclusions: Despite the absence of 2′ hydroxyls, single-stranded DNA can adopt structures that promote divalent-metal-dependent self-cleavage via an oxidative mechanism. These results suggest that an efficient DNA enzyme might be made to cleave DNA in a biological context.     

Pyrene modification leads to increased catalytic activity in minimal hammerhead ribozymes

Highly active variants of minimal hammerhead ribozymes are generated by the replacement of substantial parts of stem-loop structures with pyrene building blocks.     

Analysis of the conserved P9-G10.1 metal-binding motif in hammerhead ribozymes with an extra nucleotide inserted between A9 and G10.1 residues

Hammerhead ribozymes (Rz) have catalytically important tandem G:A pairs in the core region, and we recently demonstrated that the P9-G10.1 motif (a sheared-type G:A pair with a guanine residue on the 3' side of the adenine residue) with several flanking base pairs is sufficient for capture of divalent cations, such as Mg(2+) and Cd(2+) ions that are important to maintain full activities (Tanaka et al. J. Am. Chem. Soc. 2002, 124, 4595-4601; Tanaka et al. J. Am. Chem. Soc. 2004, 126, 744-752). We also found that mutant hammerhead ribozymes that have an additional G residue inserted between A9 and G10.1 residues (the metal-binding P9-G10.1 motif) have significant catalytic activities. In this study, we demonstrate that the hammerhead ribozymes are capable of maintaining the catalytically competent structure even when the tandem, sheared-type G:A pairs were perturbed by an insertion of an additional nucleotide, whereas the chirality of the phosphorothioate at the P9 position significantly influenced the enzymatic activity for both the natural and G-inserted ribozymes.     

Smooth solvation method for d-orbital semiempirical calculations of biological reactions. 2. Application to transphosphorylation thio effects in solution

Density-functional and semiempirical quantum methods and continuum dielectric and explicit solvation models are applied to study the role of solvation on the stabilization of native and thio-substituted transphosphorylation reactions. Extensive comparison is made between results obtained from the different methods. For the semiempirical methods, explicit solvation was treated using a hybrid quantum mechanical/molecular mechanical (QM/MM) approach and the implicit solvation was treated using a recently developed smooth solvation model implemented into a d-orbital semiempirical framework (MNDO/d-SCOSMO) within CHARMM. The different quantum and solvation methods were applied to the transesterification of a 3'-ribose,5'-methyl phosphodiester that serves as a nonenzymatic model for the self-cleavage reaction catalyzed by the hammerhead and hairpin ribozymes. Thio effects were studied for a double sulfur substitution at the nonbridging phosphoryl oxygen positions. The reaction profiles of both the native and double sulfur-substituted reactions from the MNDO/d-SCOSMO calculations were similar to the QM/MM results and consistent with the experimentally observed trends. These results underscore the need for a d-orbital semiempirical representation for phosphorus and sulfur for the study of experimentally observed thio effects in enzymatic and nonenzymatic phosphoryl transfer reactions. One of the major advantages of the present approach is that it can be applied to model chemical reactions at a significantly lower computational cost than either the density-functional calculations with implicit solvation or the semiempirical QM/MM simulations with explicit solvent.     

Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metalion-binding pockets created by the RNA fold's electrostatic field.     

Extremely high and specific activity of DNA enzymes in cells with a Philadelphia chromosome

BACKGROUND: Chronic myelogenous leukemia (CML) results from chromosome 22 translocations (the Philadelphia chromosome) that creates BCR-ABL fusion genes, which encode two abnormal mRNAs (b3a2 and b2a2). Various attempts to design antisense oligonucleotides that specifically cleave abnormal L6 BCR-ABL fusion mRNA have not been successful. Because b2a2 mRNA cannot be effectively cleaved by hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to engineer specific cleavage of this chimeric mRNA. Nonspecific effects associated with using antisense molecules make the use of such antisense molecules questionable. RESULTS: The usefulness of DNA enzymes in specifically suppressing expression of L6 BCR-ABL mRNA in mammalian cells is demonstrated. Although the efficacy of DNA enzymes with natural linkages decreased 12 hours after transfection, partially modified DNA enzymes, with either phosphorothioate or 2'-O-methyl groups at both their 5' and 3' ends, remained active for much longer times in mammalian cells. Moreover, the DNA enzyme with only 2'-O-methyl modifications was also highly specific for abnormal mRNA. CONCLUSIONS: DNA enzymes with 2'-O-methyl modifications are potentially useful as gene-inactivating agents in the treatment of diseases such as CML. In contrast to conventional antisense DNAs, some of the DNA enzymes used in this study were highly specific and cleaved only abnormal BCR-ABL mRNA.     

The pK(a) of the internucleotidic 2'-hydroxyl group in diribonucleoside (3'-->5') monophosphates

Ionization of the internucleotidic 2'-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariat-RNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pK(a) of 2'-OH in mono- and diribonucleoside (3'-->5') monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pK(a) values for internucleotidic 2'-OH of eight different diribonucleoside (3'-->5') monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pK(a) is 12.31 (+/-0.02) for ApG and 12.41 (+/-0.04) for ApA, 12.73 (+/-0.04) for GpG and 12.71 (+/-0.08) for GpA, 12.77 (+/-0.03) for CpG and 12.88 (+/-0.02) for CpA, and 12.76 (+/-0.03) for UpG and 12.70 (+/-0.03) for UpA. By comparing the pK(a)s of the respective 2'-OH of monomeric nucleoside 3'-ethyl phosphates with that of internucleotidic 2'-OH in corresponding diribonucleoside (3'-->5') monophosphates, it has been confirmed that the aglycons have no significant effect on the pK(a) values of their 2'-OH under our measurement condition, except for the internucleotidic 2'-OH of 9-adeninyl nucleotide at the 5'-end (ApA and ApG), which is more acidic by 0.3-0.4 pK(a) units.     

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site‐specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′‐branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

Repurposing Antiviral Drugs for Orthogonal RNA-Catalyzed Labeling of RNA

In vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2′,5′-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.     

In vitro selection of a novel nuclease-resistant RNA phosphodiesterase

BACKGROUND: Ribonucleotide-based enzymes (ribozymes) that cleave pathological RNAs are being developed as therapeutic agents. Chemical modification of the hammerhead ribozyme has produced nuclease-resistant catalysts that cleave targeted mRNAs in cell culture and exhibit antitumor activity in animals. Unfortunately, stabilizing modifications usually reduce the catalytic rate in vitro. An alternative to rationally designed chemical modifications of existing ribozymes is to identify novel motifs through in vitro selection of nuclease-stable sequence space. This approach is desirable because the catalysts can be optimized to function under simulated physiological conditions. RESULTS: Utilizing in vitro selection, we have identified a nuclease-stable phosphodiesterase that demonstrated optimal activity at simulated physiological conditions. The initial library of 10(14) unique molecules contained 40 randomized nucleotides with all pyrimidines in a nuclease-stabilized 2'-deoxy-2'-amino format. The selection required trans-cleaving activity and base-pairing specificity towards a resin-bound RNA substrate. Initial selective pressure was permissive, with a 30 min reaction time and 25 mM Mg(2+). Stringency of selection pressure was gradually increased until final conditions of 1 mM Mg(2+) and less than 1 min reaction times were achieved. The resulting 61-mer catalyst required the 2'-amino substitutions at selected pyrimidine positions and was stable in human serum (half-life of 16 h). CONCLUSIONS: We demonstrated that it is possible to identify completely novel, nuclease-resistant ribozymes capable of trans-cleaving target RNAs at physiologically relevant Mg(2+) concentrations. The new ribozyme motif has minimal substrate requirements, allowing for a wide range of potential RNA targets.     

An active-site guanine participates in glmS ribozyme catalysis in its protonated state

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.     

Generalized RNA-directed recombination of RNA

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.     

Nature of the chemical bond formed with the structural metal ion at the A9/G10.1 motif derived from hammerhead ribozymes

We have studied the interaction between metal ions and the metal ion-binding motif in hammerhead ribozymes, as well as the functions of the metal ion at the motif, with heteronuclear NMR spectroscopy. In this study, we employed model RNA systems which mimic the metal ion-binding motif and the altered motif. In Co(NH3)6(III) titrations, we observed large 1H and 31P chemical shift perturbations for the motif and found that outer-sphere complexation of Co(NH3)6(III) is possible for this motif. From the reinvestigation of our previous 15N chemical shift data for Cd(II) binding, in comparison with those of organometallic compounds, we conclude that Cd(II) can form an inner-sphere complex with the nucleobase in the motif. Therefore, the A9/G10.1 site was found to accept both inner-sphere and outer-sphere complexations. The Mg(II) titration for a slightly different motif from the A9/G10.1 site (G10.1-C11.1 to A10.1-U11.1) revealed that its affinity to Mg(II) was drastically reduced, although the ribozyme with this altered motif is known to retain enzymatic activities. This observation suggests that the metal ion at these motifs is not a catalytic center of hammerhead ribozymes.     

Ligand requirements for glmS ribozyme self-cleavage

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage. Herein, we demonstrate that ligand is absolutely requisite for glmS ribozyme self-cleavage activity. Furthermore, catalysis both requires and is dependent upon the acid dissociation constant (pKa) of the amine functionality of GlcN6P and related compounds. The data demonstrate that ligand is integral to catalysis, consistent with a coenzyme role for GlcN6P and illustrating an expanded capacity for biological RNA catalysis.     

Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects

The discovery of naturally occurring catalytic RNA (RNA enzymes, or ribozymes) in the 1980s immediately revised the view of RNA as a passive messenger that solely carries information from DNA to proteins. Because DNA and RNA differ only by the absence or presence of a 2'-hydroxyl group on each ribose ring of the polymer, the question of 'catalytic DNA?' arises. Although no natural DNA catalysts have been reported, since 1994 many artificial DNA enzymes, or 'deoxyribozymes', have been described. Deoxyribozymes offer insight into the mechanisms of natural and artificial ribozymes. DNA enzymes are also used as tools for in vitro and in vivo biochemistry, and they are key components of analytical sensors. This review focuses primarily on catalytic DNA for synthetic applications. Broadly defined, deoxyribozymes may have the greatest potential for catalyzing reactions in which the high selectivities of 'enzymes' are advantageous relative to traditional small-molecule catalysts. Although the scope of DNA-catalyzed synthesis is currently limited in most cases to oligonucleotide substrates, recent efforts have began to expand this frontier in promising new directions.