Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes

We demonstrated previously that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme. Moreover, the active species appeared to form dimeric structures with a common stem II (in order to distinguish monomeric forms of conventional minizymes that have low activity from our novel dimers with high-level activity, the latter very active short ribozymes were designated 'maxizymes'). The dimers can be homodimeric (with two identical binding sequences) or heterodimeric (with two different binding sequences). In the case of heterodimers, they are in equilibrium with inactive homodimers. In this study, we investigated the effects of cationic detergent, cetyltrimethylammonium bromide (CTAB), on reactions catalyzed by a variety of maxizymes. The slope of close to unity in profiles of pH versus rate demonstrated that the deprotonation was important in catalysis and that the rate-limiting chemical step was followed in these reactions. Addition of appropriate amounts of CTAB enhanced the activity of a variety of maxizymes. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. Thus, CTAB effectively enhanced the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of appropriate amounts of CTAB.     

Cleavage of an inaccessible site by the maxizyme with two independent binding arms: an alternative approach to the recruitment of RNA helicases

To overcome obstacles to target site selection, we recently created a novel hybrid ribozyme that could access any chosen site by the recruitment of intracellular RNA helicases [Warashina et al. (2001) Proc. Natl. Acad. Sci. USA 98, 5572-5577; Kawasaki et al. (2002) Nat. Biotech. 20, 376-380]. We also demonstrated previously that pol III-driven maxizymes with two substrate-binding arms that were directed against two different sites within a target mRNA formed very active heterodimers in vivo [Kuwabara, et al. (2000) Trends Biotechnol. 18, 462-468; Tanabe et al. (2001) Nature 406, 473-474]. Despite the complicated dimerization process, all the maxizymes that we tested in cultured cells had greater catalytic activity than the parental ribozymes. To investigate the action of maxizymes in cells, we designed a specific maxizyme with two substrate-binding arms that was directed against endogenously expressed LTR-luciferase chimeric mRNA, where LTR refers to the long terminal repeat of HIV-1. One substrate-binding arm of the maxizyme was designed to bind to a site within HIV-1 TAR RNA that is known to form a stable stem structure that normally prevents binding of a ribozyme. The other substrate-binding arm was directed against a relatively accessible site within the luciferase gene. As expected, the conventional ribozyme failed to cleave the TAR region in vivo because of the latter's stable secondary structure. However, to our surprise, the maxizyme cleaved the TAR region within the stem with high efficiency in vivo. The enhanced cleavage in vivo by the maxizyme might have resulted from an entropically favorable, intramolecular, second binding process that occurred during the breathing of the stem structure of the target mRNA. Importantly, our data suggest that this maxizyme technology might be used as an alternative approach to the recruitment of RNA helicases in cleaving sites previously found to be inaccessible.     

Specificity of novel allosterically trans- and cis-activated connected maxizymes that are designed to suppress BCR-ABL expression

Chronic myelogenous leukemia (CML) is associated with the presence of the Philadelphia chromosome, which is generated by the reciprocal translocation of chromosomes 9 and 22. In the case of L6 (b2a2) mRNA, it is difficult to cleave the abnormal mRNA specifically because the mRNA includes no sequences that can be cleaved efficiently by conventional hammerhead ribozymes near the BCR-ABL junction. We recently succeeded in designing a novel maxizyme, which specifically cleaves BCR-ABL fusion mRNA, as a result of the formation of a dimeric structure. As an extension of our molecular engineering of maxizymes, as well as to improve their potential utility, we examined whether an analogous conformational change could be induced within a single molecule when two maxizymes were connected via a linker sequence. An active conformation was achieved by binding of the construct to the BCR-ABL junction in trans, with part of the linker sequence then acting as an antisense modulator in cis (within the complex) to adjust the overall structure. Results of studies in vitro in the presence of cetyltrimethylammonium bromide (CTAB) (but not in its absence) suggested that a certain kind of connected maxizyme (cMzB) might be able to undergo a desired conformational change and, indeed, studies in vivo confirmed this prediction. Therefore, we successfully created a fully functional, connected maxizyme and, moreover, we found that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of CTAB.     

Working at the cutting edge: the creation of allosteric ribozymes

The appropriate folding of catalytic RNA is a prerequisite for effective catalysis. A novel ribozyme, the maxizyme, has been generated and its activity can be controlled allosterically. The maxizymes work both in vitro and in vivo indicating the potential utility of this novel class of ribozyme as a gene-inactivating agent with a biosensor function.     

Allosterically controllable maxizymes cleave mRNA with high efficiency and specificity

Ribozymes are small and versatile nucleic acids that can cleave RNA molecules at specific sites. However, because of the limited number of cleavable sequences on the target mRNA, in some cases conventional ribozymes do not have precise cleavage specificity. To overcome this problem, an allosteric version (a maxizyme) was developed that displayed activity and specificity in vivo. More than five custom-designed maxizymes have demonstrated sensor functions, which indicates that the technology might be broadly applicable in molecular biology and possibly in the clinic.     

Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications.

Hammerhead ribozymes with long antisense flanks (>50 bases) have been used successfully to inhibit replication of human immunodeficiency virus type 1 (HIV-1) in living cells. To explain their increased efficacy versus antisense controls or catalytically inactive derivatives, one can consider dissociation of the ribozyme-product complex to allow a complete catalytic cycle. In this work we investigated the dissociation of a double-stranded RNA with 56 bp in vitro. Dissociation was observed in the presence of single-stranded RNA with sequence complementarity to one of the duplex strands. A displacement reaction between RNA single strands and the duplex, but not simple dissociation, was strongly suggested by the concentration dependence of this process, the influence of additional non-complementary sequences on the single strand and by the unusually low Arrhenius activation energy. The strand displacement reaction was slow in vitro at 37 degrees C and physiological ionic strength, but was increased to k approximately 10(3)-10(4)/M/s (approximately 10(4)-fold) at higher temperatures by cetyltrimethylammonium bromide. This compound is thought to enhance non-sequence-specific association of nucleic acids in a mechanistically similar way to that in which cellular hnRNP proteins are thought to act, indicating that strand displacement can be fast and, more importantly, could be tightly regulated in vivo.     

Specific inhibition of macrophage TNF-alpha expression by in vivo ribozyme treatment.

The overproduction of the cytokine TNF-alpha is associated with inflammatory and autoimmune diseases. We have developed a means to block TNF-alpha production with ribozymes directed against TNF-alpha mRNA to selectively inhibit its production in vitro and in vivo. Following cationic lipid-mediated delivery to peritoneal murine macrophages in culture, anti-TNF-alpha ribozymes were more effective inhibitors of TNF-alpha secretion than catalytically inactive ribozyme controls. Inhibition of TNF-alpha secretion was proportional to the concentration of ribozyme administered, with an IC50 of approximately 10 nM. After i.p. injection of cationic lipid/ribozyme complexes, elicited macrophages accumulated approximately 6% of the administered ribozyme. The catalytically active ribozyme suppressed LPS-stimulated TNF-alpha secretion by approximately 50% relative to an inactive ribozyme control without inhibiting secretion of another proinflammatory cytokine produced by macrophages, IL-1alpha. Ribozyme-specific TNF-alpha mRNA degradation products were found among the mRNA extracted from macrophages following in vivo ribozyme treatment and ex vivo stimulation. Thus, catalytic ribozymes can accumulate in appropriate target cells in vivo; once in the target cell, ribozymes can be potent inhibitors of specific gene expression.     

A highly conserved domain of the maize activator transposase is involved in dimerization

Previous studies have presented indirect evidence that the transposase of the maize transposable element Activator (TPase) is active as an oligomer and forms inactive macromolecular complexes expressed in large amounts. Here, we have identified and characterized a dimerization domain at the C terminus of the protein. This domain is the most highly conserved region in the transposases of elements belonging to the Activator superfamily (hAT element superfamily) and contains a characteristic signature motif. The isolated dimerization domain forms extremely stable dimers in vitro. Interestingly, mutations in five of the six conserved residues of the signature motif do not affect in vitro dimerization, whereas mutations in other, less strictly conserved residues of the signature motif do. Loss of dimerization in vitro correlates with loss of TPase activity in vivo. As revealed by in situ immunofluorescence staining of mutant TPase proteins, the dimerization domain also is involved in forming inactive macromolecular aggregates when overexpressed, and the TPase contains one or more additional interaction functions.     

A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs.     

Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer

Lipoprotein lipase (LPL), a key enzyme in the metabolism of triglyceride-rich plasma lipoproteins, is a homodimer. Dissociation to monomers leads to loss of activity. Evidence that LPL dimers rapidly exchange subunits was demonstrated by fluorescence resonance energy transfer between lipase subunits labeled with Oregon Green and tetrametylrhodamine, respectively, and also by formation of heterodimers composed of radiolabeled and biotinylated lipase subunits captured on streptavidine-agarose. Compartmental modeling of the inactivation kinetics confirmed that rapid subunit exchange must occur. Studies of activity loss indicated the existence of a monomer that can form catalytically active dimers, but this intermediate state has not been possible to isolate and remains hypothetical. Differences in solution properties and conformation between the stable but catalytically inactive monomeric form of LPL and the active dimers were studied by static light scattering, intrinsic fluorescence, and probing with 4,4'-dianilino-1,1'-binaphtyl-5,5'-disulfonic acid and acrylamide. The catalytically inactive monomer appeared to have a more flexible and exposed structure than the dimers and to be more prone to aggregation. By limited proteolysis the conformational changes accompanying dissociation of the dimers to inactive monomers were localized mainly to the central part of the subunit, probably corresponding to the region for subunit interaction.     

In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II.

Hammerhead ribozymes were transcribed from a dsDNA template containing four random nucleotides between stems II and III, which replace the naturally occurring GAA nucleotides. In vitro selection was used to select hammerhead ribozymes capable of in cis cleavage using denaturing polyacrylamide gels for the isolation of cleaving sequences. Self-cleaving ribozymes were cloned after the first and second rounds of selection, sequenced and characterised. Only sequences containing 5'-HGAA-3', where H is A, C or U, between stems II and III were active; G was clearly not tolerated at this position. Thus, only three sequences out of the starting pool of 256 (4(4)) were active. The Michaelis-Menten parameters were determined for the in trans cleaving versions of these ribozymes and indicate that selected ribozymes are less efficient than the native sequence. We propose that the selected ribozymes accommodate the extra nucleotide as a bulge in stem II.     

Selection of intracellularly active ribozymes in mammalian cells.

Ribozymes are expected to be useful as antiviral agents and powerful tools of functional analysis of unknown gene products in vivo. For use of ribozymes in vivo, they must be fully functional in the intracellular environment. Not all ribozymes selected in vitro would be expected to work in vivo, whereas ribozymes selected in the intracellular environment should retain their function in vivo. With the eventual aim of using ribozymes as antiviral agents or biological tools in mammalian cells, we then devised a novel selection system in mammalian cells of active ribozymes by targeting at a gene for the cyclin dependent kinase inhibitor (CDKI), p16INK4a. In this system, we found that p16INK4a-knockdown cells became malignant and they formed foci. In the mammalian system, we confirmed that the selected cells harbored the active ribozyme, indicating that our positive selection systems in vivo were operational.     

Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg(2+) concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg(2+). Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.     

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.     

Chemical and enzymatic probing of effector-mediated changes in the conformation of a maxizyme

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. We designed the maxizyme in such a way that it was able to form an active core with which to capture the catalytically indispensable Mg2+ ions only in the presence of the BCR-ABL mRNA junction. In order to probe the putative conformational changes, we used a weakly alkaline solution (pH 9.2) in the presence of 25 mM Mg2+ ions to hydrolyze differentially phosphodiester bonds that were located in different environments. Phosphodiester bonds in single-stranded regions were clearly more susceptible to attack by alkali than those within a double-stranded helix. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA. Moreover, we demonstrated that the use of mild alkaline solutions to probe RNA structures is very informative.     

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.     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.     

Unfolding of in planta activity of anti-rep ribozyme in presence of a RNA silencing suppressor

Antisense RNA ribozymes have intrinsic endonucleolytic activity to effect cleavage of the target RNA. However, this activity in vivo is often controlled by the dominance of antisense or other double-stranded RNA mechanism. In this work, we demonstrate the in planta activity of a hammerhead ribozyme designed to target rep-mRNA of a phytopathogen Mungbean Yellow Mosaic India virus (MYMIV) as an antiviral agent. We also found RNA-silencing is induced on introduction of catalytically active as well as inactive ribozymes. Using RNA-silencing suppressors (RSS), we demonstrate that the endonucleolytic activity of ribozymes is a true phenomenon, even while a mutated version may demonstrate a similar down-regulation of the target RNA. This helps to ease the confusion over the action mechanism of ribozymes in vivo.     

Probing of the secondary structure of maxizymes.

The protein encoded by chimeric BCR-ABL mRNA causes chronic myelogenous leukemia (CML). We showed previously that a novel allosterically controllable ribozyme, of the type known as a maxizyme, can cleave this mRNA, with high specificity and high-level activity in vivo. In order to probe the putative conformational changes, we used a weakly alkaline solution to hydrolyze differentially phosphodiester bonds that were located in different environments. As indicated by earlier data obtained in vivo, our results demonstrated that the active conformation was achieved only in the presence of the junction within the chimeric BCR-ABL mRNA.