Short Ribozyme Research Articles

A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme.

The RNA world hypothesis suggests that chemical networks consisting of functional RNA molecules could have constructed a primitive life-like system leading a first living system. The chemical evolution scenario of RNA molecules should be consistent with the Hadean Earth environment. We have demonstrated the importance of the environment at both high temperature and high pressure, using different types of hydrothermal flow reactor systems and high-pressure equipment. In the present study, we have attempted to develop an alternative easy-to-implement method for high-pressure measurements and demonstrate that the system is applicable as an efficient research tool for high-pressure experiments at pressures up to 30 MPa. We demonstrate the usefulness of the system by detecting the high-pressure influence for the self-cleavage of avocado hammerhead ribozyme (ASBVd(−):HHR) at 45–65 °C. A kinetic analysis of the high-pressure behavior of ASBVd(−):HHR shows that the ribozyme is active at 30 MPa and its activity is sensitive to pressures between 0.1–30 MPa. The surprising finding that such a short ribozyme is effective for self-cleavage at a high pressure suggests the importance of pressure as a factor for selection of adaptable RNA molecules towards an RNA-based life-like system in the Hadean Earth environment deep in the ocean.

A combinatorial method to isolate short ribozymes from complex ribozyme libraries.

In vitro selections are the only known methods to generate catalytic RNAs (ribozymes) that do not exist in nature. Such new ribozymes are used as biochemical tools, or to address questions on early stages of life. In both cases, it is helpful to identify the shortest possible ribozymes since they are easier to deploy as a tool, and because they are more likely to have emerged in a prebiotic environment. One of our previous selection experiments led to a library containing hundreds of different ribozyme clusters that catalyze the triphosphorylation of their 5′-terminus. This selection showed that RNA systems can use the prebiotically plausible molecule cyclic trimetaphosphate as an energy source. From this selected ribozyme library, the shortest ribozyme that was previously identified had a length of 67 nucleotides. Here we describe a combinatorial method to identify short ribozymes from libraries containing many ribozymes. Using this protocol on the library of triphosphorylation ribozymes, we identified a 17-nucleotide sequence motif embedded in a 44-nucleotide pseudoknot structure. The described combinatorial approach can be used to analyze libraries obtained by different in vitro selection experiments.

Early self-reproduction, the emergence of division mechanisms in protocells

Synthetic Biology approaches are proposing model systems and providing experimental evidences that life can arise as spontaneous chemical self-assembly process where the ability to reproduce itself is an essential feature of the living system. The appearance of early cells has required an amphiphilic membrane compartment to confine molecular information against diffusion, and the ability to self-replicate the boundary layer and the genetic information. The initial spontaneous self-replication mechanisms based on thermodynamic instability would have evolved in a prebiotic and later biological catalysis. Early studies demonstrate that fatty acids spontaneously assemble into bilayer membranes, building vesicles able to grow by incorporation of free lipid molecules and divide. Early replication mechanisms may have seen inorganic molecules playing a role as the first catalysts. The emergence of a short ribozyme or short catalytic peptide may have initiated the first prebiotic membrane lipid synthesis required for vesicle growth. The evolution of early catalysts towards the simplest translation machine to deliver proteins from RNA sequences was likely to give early birth to one single enzyme controlling protocell membrane division. The cell replication process assisted by complex enzymes for lipid synthesis is the result of evolved pathways in early cells. Evolution from organic molecules to protocells and early cells, thus from chemistry to biology, may have occurred in and out of the boundary layer. Here we review recent experimental work describing membrane and vesicle division mechanisms based on chemico-physical spontaneous processes, inorganic early catalysis and enzyme based mechanisms controlling early protocell division and finally the feedback from minimal genome studies.

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2'-3' phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.

Optimization and optimality of a short ribozyme ligase that joins non-Watson-Crick base pairings.

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

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.

CTAB-mediated enrichment for active forms of novel dimeric maxizymes

We demonstrated previously that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). As a result of such a dimeric structure, heterodimeric maxizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In this case, active heterodimers are in equilibrium with inactive homodimers. Longer forms of common stem II can lead to enrichment of the active heterodimers in vitro. In this study, we investigated whether the cationic detergent CTAB, which is known to enhance strand displacement of nucleic acids, might inhibit the dimerization of maxizymes. Significantly, under all conditions examined, CTAB instead enhanced the activity of a variety of maxizymes, with the extent of enhancement depending on the conditions. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. The strand displacement activity of CTAB thus appears to enhance the conversion of alternative conformations of inactive maxizymes, with intra- and inter-molecular hydrogen bonds, to active forms. Thus, our smallest maxizyme can also be considered a potential candidate for a gene-inactivating agent in vivo, in view of the fact that various facilitators of strand displacement reactions are known to exist in vivo (indeed, a separate experiment in cell culture supported the conclusion that our smallest maxizyme is a good gene-inactivating agent). Although activities of ribozymes in vitro do not necessarily reflect their activities in vivo, our findings suggest that the activity of ribozymes in vivo can be better estimated by running ribozyme kinetics in the presence of CTAB in vitro.

TRNAVal-heterodimeric maxizymes with high potential as geneinactivating agents: simultaneous cleavage at two sites in HIV-1 Tat mRNA in cultured cells.

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNAVal-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNAVal-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNAVal-driven maxizymes tested to date have been more effective than tRNAVal-driven "standard" hammerhead ribozymes, the tRNAVal-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis.

Formation of a catalytically active dimer by tRNA(Val)-driven short ribozymes.

A minizyme is a hammerhead ribozyme with a short oligonucleotide linker instead of stem/loop II. Minizymes with low activity as monomers form active dimeric structures with a common stem. We explored the use of dimeric minizymes as gene-inactivating agents by placing minizymes under the control of a tRNA(Val) promoter. The tRNA(Val) portion of the transcript did not hinder dimerization as the tRNA-embedded minizyme formed an active dimeric structure. The cleavage activity of this minizyme that had been expressed either in vitro or in HeLa cells was almost one order of magnitude higher than that of the tRNA(Val)-embedded conventional hammerhead ribozyme. The tRNA(Val)-driven minizyme inhibited reporter gene activity (95%) whereas the tRNA(Val)-driven hammerhead ribozyme resulted in approximately 55% inhibition.

Complex formation determines the activity of ribozymes directed against potato virus Y N genomic RNA sequences

A ribozyme was synthesized against a conserved region in the RNA-dependent RNA-polymerase encoding cistron of the important plant pathogen potato virus Y (PVY). This ribozyme was shown to cleave PVY-specific RNA-transcripts efficiently in vitro, with up to 95% of the substrate RNA being cleaved within 2 h incubation at 37°C. A second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to cleave the substrates previously cleaved by the longer ribozyme. A much shorter PVY specific RNA-transcript of only 37 nucleotides (nt), however, was cleaved by this short ribozyme proving its ribozymic activity and indicating that the cleavage activity of the ribozyme is, in part, determined by the substrate involved. Analysis of cleavage reactions on non-denaturing polyacrylamide (PAA) gels indicated that incorrect basepairing, interfering with correct formation of the hammerhead structure, was likely to be responsible for the absence of detectable cleavage of the larger substrates by the short ribozyme.