Ligase Ribozyme Research Articles

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation

The acquisition of new RNA functions through evolutionary processes was essential for the diversification of RNA-based primordial biology and its subsequent transition to modern biology. However, the mechanisms by which RNAs access new functions remain unclear. Do RNA enzymes need completely new folds to support new but related functions, or is reoptimization of the active site sufficient? What are the roles of neutral and adaptive mutations in evolutionary innovation? Here, we address these questions experimentally by focusing on the evolution of substrate specificity in RNA-catalyzed RNA assembly. We use directed in vitro evolution to show that a ligase ribozyme that uses prebiotically relevant 5'-phosphorimidazole-activated substrates can be evolved to catalyze ligation with substrates that are 5'-activated with the biologically relevant triphosphate group. Interestingly, despite catalyzing a related reaction, the new ribozyme folds into a completely new structure and exhibits promiscuity by catalyzing RNA ligation with both triphosphate and phosphorimidazole-activated substrates. Although distinct in sequence and structure, the parent phosphorimidazolide ligase and the evolved triphosphate ligase ribozymes can be connected by a series of point mutations where the intermediate sequences retain at least some ligase activity. The existence of a quasi-neutral pathway between these distinct ligase ribozymes suggests that neutral drift is sufficient to enable the acquisition of new substrate specificity, thereby providing opportunities for subsequent adaptive optimization. The transition from RNA-catalyzed RNA assembly using phosphorimidazole-activated substrates to triphosphate-activated substrates may have foreshadowed the later evolution of the protein enzymes that use monomeric triphosphates (nucleoside triphosphates, NTPs) for RNA synthesis.

A cis-acting ligase ribozyme generates circular RNA in vitro for ectopic protein functioning

Delivering synthetic protein-coding RNA bypassing the DNA stage for ectopic protein functioning is a novel therapeutic strategy. Joining the linear RNA head-to-tail covalently could be a state-of-the-art strategy for functioning longer. Here we enroll a cis-acting ligase ribozyme (RzL) to generate circular RNA (circRNA) in vitro for ectopic protein expression. The RNA circularization is confirmed by masking the 5’ phosphate group, resisting exonuclease RNase R digestion, failing for further tailing, and sequencing the RT-PCR products of the joined region. Interestingly, one internal ribosome entry site (IRES) renders circRNA translation competent, but two IRES in cis, not trans, hamper the translation. The circRNA with highly potent in translation is conferred for antiviral functioning. Accompanying specific guided RNA, a circRNA expressing ribonuclease Cas13 shows excellent potential against the corresponding RNA virus, further extending circRNA functioning in its growing list of applications.

Development of Allosteric Ribozymes for ATP and l-Histidine Based on the R3C Ligase Ribozyme.

During the evolution of the RNA, short RNAs are thought to have joined together to form long RNAs, enhancing their function as ribozymes. Previously, the artificial R3C ligase ribozyme (73 nucleotides) was successfully reduced to 46 nucleotides; however, its activity decreased significantly. Therefore, we aimed to develop allosteric ribozymes, whose activities could be regulated by effector compounds, based on the reduced R3C ligase ribozyme (R3C-A). Among the variants prepared by fusing an ATP-binding aptamer RNA with R3C-A, one mutant showed increased ligation activity in an ATP-dependent manner. Melting temperature measurements of the two RNA mutants suggested that the region around the aptamer site was stabilized by the addition of ATP. This resulted in a suitable conformation for the reaction at the ligation site. Another ribozyme was prepared by fusing R3C-A with a l-histidine-binding aptamer RNA, and the ligase activity increased with increasing l-histidine concentrations. Both ATP and l-histidine play prominent roles in current molecular biology and the interaction of RNAs and these molecules could be a key step in the evolution of the world of RNAs. Our results suggest promise in the development of general allosteric ribozymes that are independent of the type of effector molecule and provide important clues to the evolution of the RNA world.

Natural soda lakes provide compatible conditions for RNA and membrane function that could have enabled the origin of life.

The origin of life likely occurred within environments that concentrated cellular precursors and enabled their co-assembly into cells. Soda lakes (those dominated by Na+ ions and carbonate species) can concentrate precursors of RNA and membranes, such as phosphate, cyanide, and fatty acids. Subsequent assembly of RNA and membranes into cells is a long-standing problem because RNA function requires divalent cations, e.g. Mg2+, but Mg2+ disrupts fatty acid membranes. The low solubility of Mg-containing carbonates limits soda lakes to moderate Mg2+ concentrations (∼1 mM), so we investigated whether both RNAs and membranes function within these lakes. We collected water from Last Chance Lake and Goodenough Lake in Canada. Because we sampled after seasonal evaporation, the lake water contained ∼1 M Na+ and ∼1 mM Mg2+ near pH 10. In the laboratory, nonenzymatic, RNA-templated polymerization of 2-aminoimidazole-activated ribonucleotides occurred at comparable rates in lake water and standard laboratory conditions (50 mM MgCl2, pH 8). Additionally, we found that a ligase ribozyme that uses oligonucleotide substrates activated with 2-aminoimidazole was active in lake water after adjusting pH from ∼10 to 9. We also observed that decanoic acid and decanol assembled into vesicles in a dilute solution that resembled lake water after seasonal rains, and that those vesicles retained encapsulated solutes despite salt-induced flocculation when the external solution was replaced with dry-season lake water. By identifying compatible conditions for nonenzymatic and ribozyme-catalyzed RNA assembly, and for encapsulation by membranes, our results suggest that soda lakes could have enabled cellular life to emerge on Earth, and perhaps elsewhere.

Molecular Anatomy of the Class I Ligase Ribozyme for Elucidation of the Activity-Generating Unit.

The class I ligase ribozyme consists of 121 nucleotides and shows a high catalytic rate comparable to that found in natural proteinaceous polymerases. In this study, we aimed to identify the smaller active unit of the class I ligase ribozyme comprising ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Based on the three-dimensional structure of the class I ligase ribozyme, mutants were prepared and their ligation activities were analyzed. Sufficient ligation activity was maintained even when shortening to 94 nucleotides. However, because it would be difficult to approach the target of ~50 nucleotides by removing only the partial structure, the class I ligase ribozyme was then split into two molecules. The ligation activity was maintained even when splitting into two molecules of 55 and 39 nucleotides. Using a system with similar split ribozymes, we analyzed the ligation activity of mutants C30, C47, and A71, which have been previously identified as the positions that contribute to catalytic activity, and discussed the structural basis of the activity of these bases. Our findings suggest the rationale for the class I ligase ribozyme's assembling from multiple fragments that would be achievable with prebiotic synthesis.

Ribozyme activity modulates the physical properties of RNA-peptide coacervates.

Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype-phenotype linkage.

RNA ligase ribozymes with a small catalytic core

Catalytic RNAs, or ribozymes, catalyze diverse chemical reactions that could have sustained primordial life in the hypothetical RNA world. Many natural ribozymes and laboratory evolved ribozymes exhibit efficient catalysis mediated by elaborate catalytic cores within complex tertiary structures. However, such complex RNA structures and sequences are unlikely to have emerged by chance during the earliest phase of chemical evolution. Here, we explored simple and small ribozyme motifs capable of ligating two RNA fragments in a template-directed fashion (ligase ribozymes). One-round selection of small ligase ribozymes followed by deep sequencing revealed a ligase ribozyme motif comprising a three-nucleotide loop opposite to the ligation junction. The observed ligation was magnesium(II) dependent and appears to form a 2′–5′ phosphodiester linkage. The fact that such a small RNA motif can function as a catalyst supports a scenario in which RNA or other primordial nucleic acids played a central role in chemical evolution of life.

Minimal RNA self-reproduction discovered from a random pool of oligomers.

The emergence of RNA self-reproduction from prebiotic components would have been crucial in developing a genetic system during the origins of life. However, all known self-reproducing RNA molecules are complex ribozymes, and how they could have arisen from abiotic materials remains unclear. Therefore, it has been proposed that the first self-reproducing RNA may have been short oligomers that assemble their components as templates. Here, we sought such minimal RNA self-reproduction in prebiotically accessible short random RNA pools that undergo spontaneous ligation and recombination. By examining enriched RNA families with common motifs, we identified a 20-nucleotide (nt) RNA variant that self-reproduces via template-directed ligation of two 10 nt oligonucleotides. The RNA oligomer contains a 2'-5' phosphodiester bond, which typically forms during prebiotically plausible RNA synthesis. This non-canonical linkage helps prevent the formation of inactive complexes between self-complementary oligomers while decreasing the ligation efficiency. The system appears to possess an autocatalytic property consistent with exponential self-reproduction despite the limitation of forming a ternary complex of the template and two substrates, similar to the behavior of a much larger ligase ribozyme. Such a minimal, ribozyme-independent RNA self-reproduction may represent the first step in the emergence of an RNA-based genetic system from primordial components. Simultaneously, our examination of random RNA pools highlights the likelihood that complex species interactions were necessary to initiate RNA reproduction.

Acquisition of Dual Ribozyme-Functions in Nonfunctional Short Hairpin RNAs through Kissing-Loop Interactions.

The acquisition of functions via the elongation of nucleotides is an important factor in the development of the RNA world. In our previous study, we found that the introduction of complementary seven-membered kissing loops into inactive R3C ligase ribozymes revived their ligation activity. In this study, we applied the kissing complex formation-induced rearrangement of RNAs to two nonfunctional RNAs by introducing complementary seven-membered loops into each of them. By combining these two forms of RNAs, the ligase activity (derived from the R3C ligase ribozyme) as well as cleavage activity (derived from the hammerhead ribozyme) was obtained. Thus, effective RNA evolution toward the formation of a life system may require the achievement of “multiple” functions via kissing-loop interactions, as indicated in this study. Our results point toward the versatility of kissing-loop interactions in the evolution of RNA, i.e., two small nonfunctional RNAs can gain dual functions via a kissing-loop interaction.

Experimental exploration of a ribozyme neutral network using evolutionary algorithm and deep learning

A neutral network connects all genotypes with equivalent phenotypes in a fitness landscape and plays an important role in the mutational robustness and evolvability of biomolecules. In contrast to earlier theoretical works, evidence of large neutral networks has been lacking in recent experimental studies of fitness landscapes. This suggests that evolution could be constrained globally. Here, we demonstrate that a deep learning-guided evolutionary algorithm can efficiently identify neutral genotypes within the sequence space of an RNA ligase ribozyme. Furthermore, we measure the activities of all 216 variants connecting two active ribozymes that differ by 16 mutations and analyze mutational interactions (epistasis) up to the 16th order. We discover an extensive network of neutral paths linking the two genotypes and reveal that these paths might be predicted using only information from lower-order interactions. Our experimental evaluation of over 120,000 ribozyme sequences provides important empirical evidence that neutral networks can increase the accessibility and predictability of the fitness landscape.

Assembly of a Ribozyme Ligase from Short Oligomers by Nonenzymatic Ligation.

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

Systematic minimization of RNA ligase ribozyme through large-scale design-synthesis-sequence cycles.

Template-directed RNA ligation catalyzed by an RNA enzyme (ribozyme) is a plausible and important reaction that could have been involved in transferring genetic information during prebiotic evolution. Laboratory evolution experiments have yielded several classes of ligase ribozymes, but their minimal sequence requirements remain largely unexplored. Because selection experiments strongly favor highly active sequences, less active but smaller catalytic motifs may have been overlooked in these experiments. We used large-scale DNA synthesis and high-throughput ribozyme assay enabled by deep sequencing to systematically minimize a previously laboratory-evolved ligase ribozyme. After designing and evaluating >10 000 sequences, we identified catalytic cores as small as 18 contiguous bases that catalyze template-directed regiospecific RNA ligation. The fact that such a short sequence can catalyze this critical reaction suggests that similarly simple or even simpler motifs may populate the RNA sequence space which could have been accessible to the prebiotic ribozymes.

Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation

The formation of a kissing-loop through the introduction of complementary 7-membered loops is known to dramatically increase the activity of truncated R3C ligase ribozymes that otherwise display reduced activity. Restoration of activity is thought to result from kissing complex formation-induced rearrangement of two molecules with complementary loops. By combining two types of R3C ligase ribozyme mutants, <A> and <hairpin-ΔU>, the influence of loop composition on ligation activity was investigated. Substrate ligation occurred in <hairpin-ΔU>, but not in <A>, despite the absence of a substrate-binding site in <hairpin-ΔU>. Loop-loop interactions of <A>- and <hairpin-ΔU>-variants with complementary 6-membered loops also resulted in proper kissing-complex formation-induced substrate ligation. However, heterogeneous combinations of 7- and 6-membered loops, and/or of 6- and 5-membered loops had distinct results that depended upon the sequence and bulged nucleotides of the loop regions. These differences suggest that both thermodynamic and kinetic controls act upon the kissing-loop interaction-mediated rearrangement of the shortened trans-R3C ribozymes.

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life.

R3C ligase ribozyme catalyzes the nucleophilic attack by a 3′-hydroxyl on a 5′-α-phosphorus of triphosphates to form a 3′-5′-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a “switch” to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world.

Prebiotic Factors Influencing the Activity of a Ligase Ribozyme.

An RNA-lipid origin of life scenario provides a plausible route for compartmentalized replication of an informational polymer and subsequent division of the container. However, a full narrative to form such RNA protocells implies that catalytic RNA molecules, called ribozymes, can operate in the presence of self-assembled vesicles composed of prebiotically relevant constituents, such as fatty acids. Hereby, we subjected a newly engineered truncated variant of the L1 ligase ribozyme, named tL1, to various environmental conditions that may have prevailed on the early Earth with the objective to find a set of control parameters enabling both tL1-catalyzed ligation and formation of stable myristoleic acid (MA) vesicles. The separate and concurrent effects of temperature, concentrations of Mg2+, MA, polyethylene glycol and various solutes were investigated. The most favorable condition tested consists of 100 mM NaCl, 1 mM Mg2+, 5 mM MA, and 4 °C temperature, whereas the addition of Mg2+-chelating solutes, such as citrate, tRNAs, aspartic acid, and nucleoside triphosphates severely inhibits the reaction. These results further solidify the RNA-lipid world hypothesis and stress the importance of using a systems chemistry approach whereby a wide range of prebiotic factors interfacing with ribozymes are considered.

Characterization of an RNA receptor motif that recognizes a GCGA tetraloop.

Tertiary interactions between a new RNA motif and RNA tetraloops were analyzed to determine whether this new motif shows preference for a GCGA tetraloop. In the structural context of a ligase ribozyme, this motif discriminated GCGA loop from 3 other tetraloops. The affinity between the GCGA loop and its receptor is strong enough to carry out the ribozyme activity.

Handmade microfluidic device for biochemical applications in emulsion

A simple, inexpensive flow-focusing device has been developed to make uniform droplets for biochemical reactions, such as in vitro transcription and cell-free protein synthesis. The device was fabricated from commercially available components without special equipment. Using the emulsion droplets formed by the device, a class I ligase ribozyme, bcI 23, was successfully synthesized from DNA attached to magnetic microbeads by T7 RNA polymerase. It was also ligated with an RNA substrate on the same microbeads, and detected using flow cytometry with a fluorescent probe. In addition, a single-chain derivative of the lambda Cro protein was expressed using an Escherichia coli cell-free protein synthesis system in emulsion, which was prepared using the flow-focusing device. In both emulsified reactions, usage of the flow-focusing device was able to greatly reduce the coefficient of variation for the amount of RNA or protein displayed on the microbeads, demonstrating the device is advantageous for quantitative analysis in high-throughput screening.

Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions.

In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.