Improved polymerase ribozyme efficiency on hydrophobic assemblies

During an early step in the evolution of life, RNA served both as genome and as catalyst, according to the RNA world hypothesis. For self-replication, the RNA organisms must have contained an RNA that catalyzes RNA polymerization. As a first step toward recapitulating an RNA world in the laboratory, a polymerase ribozyme was generated previously by in vitro evolution and design. However, the efficiency of this ribozyme is about 100-fold too low for self-replication because of a low affinity of the ribozyme to its primer/template substrate. To improve the substrate interactions by colocalizing ribozyme and substrate on micelles, we attached hydrophobic anchors to both RNAs. We show here that the hydrophobic anchors led to aggregates with the expected size of the corresponding micelles. The micelle formation increased the polymerization yield of full-length products by 3- to 20-fold, depending on substrates and reaction conditions. With the best-characterized substrate, the improvement in polymerization efficiency was primarily due to reduced sequence-specific stalling on partially extended substrates. We discuss how, during the origin of life, micellar ribozyme aggregates could have acted as precursors to membrane-encapsulated life forms.     

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world.     

Arginine cofactors on the polymerase ribozyme

The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.     

Self-Replication Reactions Dependent on Tertiary Interaction Motifs in an RNA Ligase Ribozyme

RNA can function both as an informational molecule and as a catalyst in living organisms. This duality is the premise of the RNA world hypothesis. However, one flaw in the hypothesis that RNA was the most essential molecule in primitive life is that no RNA self-replicating system has been found in nature. To verify whether RNA has the potential for self-replication, we constructed a new RNA self-assembling ribozyme that could have conducted an evolvable RNA self-replication reaction. The artificially designed, in vitro selected ligase ribozyme was employed as a prototype for a self-assembling ribozyme. The ribozyme is composed of two RNA fragments (form R1·Z1) that recognize another R1·Z1 molecule as their substrate and perform the high turnover ligation reaction via two RNA tertiary interaction motifs. Furthermore, the substrate recognition of R1·Z1 is tolerant of mutations, generating diversity in the corresponding RNA self-replicating network. Thus, we propose that our system implies the significance of RNA tertiary motifs in the early RNA molecular evolution of the RNA world.     

A Faster Triphosphorylation Ribozyme

In support of the RNA world hypothesis, previous studies identified trimetaphosphate (Tmp) as a plausible energy source for RNA world organisms. In one of these studies, catalytic RNAs (ribozymes) that catalyze the triphosphorylation of RNA 5''-hydroxyl groups using Tmp were obtained by in vitro selection. One ribozyme (TPR1) was analyzed in more detail. TPR1 catalyzes the triphosphorylation reaction to a rate of 0.013 min^-1 under selection conditions (50 mM Tmp, 100 mM MgCl₂, 22°C). To identify a triphosphorylation ribozyme that catalyzes faster triphosphorylation, and possibly learn about its secondary structure TPR1 was subjected to a doped selection. The resulting ribozyme, TPR1e, contains seven mutations relative to TPR1, displays a previously unidentified duplex that constrains the ribozyme''s structure, and reacts at a 24-fold faster rate than the parent ribozyme. Under optimal conditions (150 mM Tmp, 650 mM MgCl₂, 40°C), the triphosphorylation rate of TRP1e reaches 6.8 min^-1.     

New ligase-derived RNA polymerase ribozymes

The search is underway for a catalytic RNA molecule capable of self-replication. Finding such a ribozyme would lend crucial support to the RNA World hypothesis, which holds that very early life-forms relied on RNA for both replicating and storing genetic information. We previously reported an RNA polymerase isolated from a pool of variants of an existing RNA ligase ribozyme. Here we report eight additional ligase-derived polymerase ribozymes isolated from this pool. Because each of them is a new potential starting point for further in vitro evolution and engineering, together they substantially enrich the set of candidates from which an RNA replicase ribozyme might eventually emerge.     

Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment

Background

The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis.

Results

The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay.

Conclusion

These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization.

     

Dinucleotide repeat expansion catalyzed by bacteriophage T4 DNA polymerase in vitro

DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments.     

Recognition of nucleoside triphosphates during RNA-catalyzed primer extension

In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K(m) and a higher k(cat), suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the gamma-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k(cat) of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.     

Structural insights into mechanisms of catalysis and inhibition in Norwalk virus polymerase

Crystal structures of Norwalk virus polymerase bound to an RNA primer-template duplex and either the natural substrate CTP or the inhibitor 5-nitrocytidine triphosphate have been determined to 1.8A resolution. These structures reveal a closed conformation of the polymerase that differs significantly from previously determined open structures of calicivirus and picornavirus polymerases. These closed complexes are trapped immediately prior to the nucleotidyl transfer reaction, with the triphosphate group of the nucleotide bound to two manganese ions at the active site, poised for reaction to the 3'-hydroxyl group of the RNA primer. The positioning of the 5-nitrocytidine triphosphate nitro group between the alpha-phosphate and the 3'-hydroxyl group of the primer suggests a novel, general approach for the design of antiviral compounds mimicking natural nucleosides and nucleotides.     

The three-dimensional architecture of the class I ligase ribozyme

The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.     

Nucleotide synthetase ribozymes may have emerged first in the RNA world

Though the "RNA world" hypothesis has gained a central role in ideas concerning the origin of life, the scenario concerning its emergence remains uncertain. It has been speculated that the first scene may have been the emergence of a template-dependent RNA synthetase ribozyme, which catalyzed its own replication: thus, "RNA replicase." However, the speculation remains uncertain, primarily because of the large sequence length requirement of such a replicase and the lack of a convincing mechanism to ensure its self-favoring features. Instead, we propose a nucleotide synthetase ribozyme as an alternative candidate, especially considering recent experimental evidence suggesting the possibility of effective nonenzymatic template-directed synthesis of RNA. A computer simulation was conducted to support our proposal. The conditions for the emergence of the nucleotide synthetase ribozyme are discussed, based on dynamic analysis on a computer. We suggest the template-dependent RNA synthetase ribozyme emerged later, perhaps after the emergence of protocells.     

The origin of life: RNA and protein co-evolution on the ancient Earth

How life emerged from simple non-life chemicals on the ancient Earth is one of the greatest mysteries in biology. The gene expression system of extant life is based on the interdependence between multiple molecular species (DNA, RNA, and proteins). While DNA is mainly used as genetic material and proteins as functional molecules in modern biology, RNA serves as both genetic material and enzymes (ribozymes). Thus, the evolution of life may have begun with the birth of a ribozyme that replicated itself (the RNA world hypothesis), and proteins and DNA joined later. However, the complete self-replication of ribozymes from monomeric substrates has not yet been demonstrated experimentally, due to their limited activity and stability. In contrast, peptides are more chemically stable and are considered to have existed on the ancient Earth, leading to the hypothesis of RNA–peptide co-evolution from the very beginning. Our group and collaborators recently demonstrated that (1) peptides with both hydrophobic and cationic moieties (e.g., KKVVVVVV) form β-amyloid aggregates that adsorb RNA and enhance RNA synthesis by an artificial RNA polymerase ribozyme and (2) a simple peptide with only seven amino acid types (especially rich in valine and lysine) can fold into the ancient β-barrel conserved in various enzymes, including the core of cellular RNA polymerases. These findings, together with recent reports from other groups, suggest that simple prebiotic peptides could have supported the ancient RNA-based replication system, gradually folded into RNA-binding proteins, and eventually evolved into complex proteins like RNA polymerase.     

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3',5'-phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA-protein world.     

Exiting an RNA world

The RNA world hypothesis gains support from the in vitro evolution of a bifunctional ribozyme that can recognize an activated glutaminyl ester and subsequently aminoacylate a tRNA molecule.     

Design and development of a catalytic ribonucleoprotein

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.     

A ribozyme that triphosphorylates RNA 5′-hydroxyl groups

The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5′-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min⁻¹ under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5′-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source.     

Selection of an improved RNA polymerase ribozyme with superior extension and fidelity

Our current understanding of biology suggests that early life relied predominantly on RNA for catalysis and replication. Here, we report the isolation of an RNA polymerase ribozyme called B6.61 that exhibits superior extension and fidelity relative to its progenitor, the Round-18 polymerase. The B6.61 polymerase was selected from a mutagenized pool containing approximately 9 x 10(14) sequence variants through the use of a novel large-scale in vitro compartmentalization system. B6.61 polymerized all tested primer-template (PT) complexes faster than the Round-18 variant. For one PT, B6.61 exhibited dramatically faster elongation past one full helical turn and incorporated at least 20 nucleotides of sequence, setting a new extension record for an RNA polymerase ribozyme. The increased efficiency of the B6.61 construct was related to improvements in fidelity, with the new variant incorporating less incorrect wobble base pairs than its parent. This new polymerase demonstrates the feasibility of evolving an artificial RNA replicase ribozyme in the foreseeable future.