Qβ Replicase Research Articles

Rapid and sensitive detection ofChlamydia trachomatisusing a ligatable binary RNA probe and Qβ replicase

A simple assay format was developed for the direct detection ofC. trachomatisrRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 104targets) was suppressed by blocking sequences in the 5′ MDV reporter probe fragment complementary to the 3′ fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid–selection–amplification protocol, yielded a low level of assay background which was reduced to <2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 103molecules of 23S rRNA (>95% responding) and could detect a single elementary body (EB) ofChlamydia trachomatisor 1–10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 105-fold range, permitting the determination of target level within an order of magnitude. The assay showed 109-fold discrimination overChlamydia pneumonae(TWAR) rRNA. High levels of culturedC. albicans,E. coli,S. aureus, orN. gonorrhoeaehad no detectable effect on assay background or the ability to detect a single elementary body.

Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qβ replicase efficiently binds small RNAs by recognizing pyrimidine residues

Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qβ was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5′-d(TTTTTCC) was 5′-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qβ replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16°C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qβ replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qβ replicase does not require a 2′ hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A·T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qβ replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows detection of RNA amplification by Qβ replicase in real-time.

Translational activation in coliphage qβ: on a polycistronic messenger RNA, repression of one gene can activate translation of another

We present evidence for translational activation of the Qβ coliphage maturation cistron, mediated by the presence of Qβ replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qβ maturation gene remains translationally “off” by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched “on” when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qβ replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Recognition of bacteriophage Qβ plus strand RNA as a template by Qβ replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor

RNA-protein interactions between bacteriophage Qβ plus strand RNA and the components of the Qβ replicase system were studied by deletion analysis. Internal, 5′-terminal and 3′-terminal deletions were assayed for template activity with replicase in vitro. Of the two internal binding sites previously described for replicase, we found that the S-site (map position 1247 to 1346) could be deleted without any significant effect on template activity, whereas deletion of the M-site (map position 2545 to 2867) resulted in a strong inactivation and a high salt sensitivity of the residual activity. Binding complexes of the deletion mutant RNAs with the different proteins involved in Qβ RNA replication were analysed by electron microscopy. The formation of looped complex structures, previously reported and explained as simultaneous interactions with replicase at the S and the M-site, was abolished by deleting the S-site but, surprisingly, not by deleting the M-site. The same types of complexes observed with replicase were also formed with purified protein S1 (the α subunit of replicase), suggesting that these internal interactions with Qβ RNA are mediated by the S1 protein. The Qβ host factor, a protein required for the template activity of the Qβ plus strand, was reported earlier to form similar complexes by binding to the S and M-sites (or adjacent sites) and in addition to the 3′-end, resulting in double-looped structures. The patterns of looped complexes observed with the deletion mutant RNAs suggest that the binding of host factor might not involve the S and M-sites themselves but adjacent downstream sites. An additional internal host factor interaction near map position 2300 was detected with several mutant RNAs. Qβ RNA molecules with 3′-truncations formed 3′-terminal loops with similar efficiency as wild-type RNA, indicating that recognition of the 3′-end by host factor is not dependent on a specific 3′-terminal base sequence.

A theory of evolution that includes prebiotic self-organization and episodic species formation

A theory has been proposed that encompasses pre-replication changes in RNA synthesis and non-gradual variant formation, in addition to competitive replication. Using a fundamental theorem of natural selection and maximum principle scaled to nucleotide condensation, evolution in vitro was demonstrated to maximally damp both kinetic and thermodynamic forces driving this reaction, from its pre-replication stage. This led to the finding that evolution follows a path of least action. These principles form the framework for a general theory of evolution, whose scope extends beyond evolution modeled by synthesis of non interacting RNA molecules. It applies, in particular, to standard processes, such as competitive crystallization. In calculations simulating de novo formation of self-replicating RNA molecules in the Qβ replicase system, spontaneous changes in strand secondary structure promoted the transition from random copolymerization to template-directed polymerization. This finding indicates selection preceded genome self-propagation. Non-gradual species formation was attributed to the presence of heterogeneous thermodynamic forces. Growth unconstrained by competition follows mutation to a variant able to utilize a free energy source alien to its progenitors. Evolution in a heterogeneous system can, therefore, exhibit discontinuous rates of species formation and spawn new species populations. Natural selection among competing self-propagators thus gives way to a principle of wider scope stating that evolution optimally damps the physicochemical forces causing change within an evolving system.

Why is the polymerase chain reaction resistant to in vitro evolution?

A variety of methods have been developed to amplify DNA and RNA. These methods vary in their susceptibility to evolve new molecular species differing from the starting template. PCR is exceptionally resistant to in vitro evolution, whereas methods such as Qβ replicase and 3SR are much less robust. This paper develops some simple mathematical models which suggest that PCR is resistant to in vitro evolution because the reaction controls replication in discrete cycles: fast replication is of little advantage during PCR because the reaction limits fast replicators as well as slow ones to a single copy per cycle. In contrast, continuous (isothermal) reactions, as in the Qβ replicase reaction, favor fast replicators. The advantage of fast replication is compounded in continuous reactions, because a fast replicator can complete many generations of replication during the time it takes a slow replicator to complete one generation. These models suggest that continuous amplication protocols will never achieve the robustness against in vitro evolution observed with PCR.

Real-Time Fluorescence Detection of RNA Amplified by Qβ Replicase

Amplification of RNA probes by Qβ replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Qβ amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 × 10 11 RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.

Applications of DNA amplification techniques in veterinary diagnostics.

An overview of the principles of the polymerase chain reaction, ligase chain reaction, self-sustained sequence replication and Qβ replicase is given. The application of these methods for the diagnosis of veterinary infectious and hereditary diseases as well as for other diagnostic purposes is discussed and comprehensive tables of reported assays are provided. Specific areas where these DNA-based amplification methods provide substantial advantages over traditional approaches are also highlighted. With regard to PCR-based assays for the detection of viral pathogens, this article is an update of a previous review by Belák and Ballagi-Pordány (1993).

The Mutant Distribution of an RNA Species Replicated by Qβ Replicase

The mutant spectrum of an RNA species that is replicated by Qβ replicase, MNV-11, was investigated by retrotranscribing the RNA to DNA and cloning it into plasmids. The sequences of several cDNA clones of MNV-11 populations amplified by Qβ replicase under various conditions were determined and compared. A surprisingly broad mutant distribution was found: the consensus sequence never made up more than 40% of the total population and was accompanied by many mutants. Most mutants had several base exchanges, insertions and/or deletions; up to nine of the total 86 nucleotides were changed. The mutants found had replication rates comparable to that of the wild-type and were thus enriched in the population by selection forces. When the growth conditions were changed, the mutant distribution centre was shifted. The published consensus sequence of MNV-11 did not have the highest growth rate of the mutants, but was rather the best adapted to the various selection forces governing the growth phases the replicating RNA went through, i.e. it had found an optimal compromise between the rates of overall replication, enzyme binding and double strand formation.f2f2Abbreviations used: TGGE, temperature gradient gel electrophoresis; HD, Hamming distance.

Stability of replicative form and fitness among RNA variants transcribed by Qbeta replicase.

Stability in the replicative form of RNA molecules transcribed by Qbeta replicase was demonstrated to provide a sequence-dependent indicator of their fitness. This follows from the finding that replication rates reported for 17 RNA species (genome length, 77-370 nucleotides) correlate with the self-interaction free energy of these self-annealed strands. Formation of double-stranded molecules during replication conversely decreased with self-interaction free energy. H-bond formation between self-complementary segments of folded RNA molecules plainly produces a potential energy barrier opposing the transition to a double-stranded, non-replicating form. Melting point temperature and resistance of RNA synthesis to elevated salt levels among three variants also increased with strand configuration free energy. Genome-based estimates of fitness in other self-replicating RNA species were therefore possible. Once a link between the kinetic parameters of replication and base sequence of these RNA species is adequately established, estimates of fitness can be dissociated from survival states following evolution, and Darwin's fundamental precept, 'survival of the fittest,' could be appraised as an experimentally testable hypothesis.

Viral Qβ RNA as a high expression vector for mRNA translation in a cell-free system

Dihydrofolate reductase (DHFR) mRNA was inserted into Qβ phage RNA instead of its coat protein cistron. Translation of this recombinant mRNA in the Escherichia coli cell-free system resulted in the synthesis of DHFR, which was two orders of magnitude higher than that in the case of translation of the control DHFR mRNA. Additionally, it resulted in a significantly enhanced synthesis of Qβ replicase as compared with its synthesis when the original Qβ RNA was used.

Enzymatic RNA Replication in Self-Reproducing Vesicles: An Approach to a Minimal Cell

The replication of a RNA template catalyzed by Qβ replicase was obtained in oleic acid/oleate vesicles simultaneously with the self-reproduction of the vesicles themselves. This was accomplished by entrapping the enzyme Qβ replicase, the RNA template, and the ribonucleotides ATP, CTP, GTP, and UTP inside the vesicles. The water-insoluble oleic anhydride was then added externally. It binds to the vesicle bilayer where it is catalytically hydrolyzed yielding the carboxylate surfactant in situ, which then brings about growth and reproduction of the vesicles themselves. This experiment is presented as a first approach to a synthetic minimal cell, in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously.

Replicable RNA Vectors: Prospects for Cell-free Gene Amplification, Expression, and Cloning

Publisher Summary This chapter discusses the possibility of utilizing the reproductive system of one of the positive-strand RNA viruses—Qβ bacteriophage, which infects Escherichia coli cells—for the amplification, expression, and cloning of foreign genes in vitro . This virus encodes a unique RNA replicase. In contrast to RNA replicases of other viruses, Qβ replicase can easily be isolated in preparative amounts and is long-lived under cell-free conditions. Qβ replicase is much more efficient in cell-free reactions than other systems utilized for the amplification of nucleic acids. It can produce as many as 10 12 copies of RNA template in less than 30 minutes of incubation. The inability of Qβ replicase to amplify exponentially heterologous templates, including the genomic RNAs of related bacteriophages, has led to a wide-spread belief in the strict template specificity of this enzyme. At the same time, Qβ replicase does amplify exponentially numerous unrelated RNA species called “RQ RNAs.”

A Study on the Function of the Glycine Residue in the YGDD Motif of the RNA-Dependent RNA Polymerase β-Subunit from RNA Coliphage Qβ1

Q beta replicases in which the Gly residue of the beta-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg(2+)-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Q beta phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.

Enzymatic RNA synthesis in self‐reproducing vesicles: An approach to the construction of a minimal synthetic cell

AbstractOur approach towards “core‐and‐shell self‐reproduction” is reviewed here. With this term, we indicate a process by which the shell‐reproduction of a spherically bounded system (micelles or vesicles) proceeds simultaneously with the replication of nucleic acid which is hosted inside the micelles or vesicles. The realization of such a process is seen as a step towards the construction of a synthetic cell model. Two chemical systems are examined here, one based on the polynucleotide phosphorylase‐catalyzed synthesis of poly(A) starting from ADP; and the other based on the enzyme Qβ replicase, which is able to catalyze the synthesis of a RNA template. In both cases, production of RNA macromolecules proceeds simultaneously with self‐reproduction of the oleic acid/oleate vesicles. One still open question is the determination of the redistribution of the guest macromolecular components during the reproduction of the shell. Within these limits, it is argued that this work offers a system which goes beyond the two approaches to self‐replication presented until now in the literature, namely the template self‐reproduction of linear sequences of oligonucleotides and the autopoietic shell reproduction of micelles and vesicles.

Construction of nuclease resistant ribonucleotides by molecular evolution.

The molecular evolution by replicase (RNA-dependent RNA polymerase) was attempted for generating new nuclease-resistant RNAs, which realizes a cell free Darwinian molecular evolution. The Qβ replicase replicates genome RNA of Qβ virus, and additionally it can also synthesize other small RNAs using “minivaliant” as a template. With the aid of Spiegelman's serial trasfer method, which is a step by step molecular evolution system in a laboratory by RNA proliferation with frequent mutation using Qβ replicase, high concentration ribonuclease T2-resistant fragments of RNA could he obtained.

Different Mechanisms of Recognition of Bacteriophage Qβ Plus and Minus Strand RNAs by Qβ Replicase

Our earlier work on the recognition of Qβ plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3′-end, i.e. the site of initiation of synthesis. Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity. In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP. A variant plus strand RNA containing a 490 nucleotide duplication near the 5′-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3′-end of the RNA. Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase. This suggests that the role of the host factor on the plus strand template is to bring the 3′-end into the proximity of the S-site/M-site domain, where replicase can initiate on it. In contrast, the 3′-end of the minus strand appears to be directly available to the enzyme.

Structure of the Maize Mitochondrial Replicon RNA b and Its Relationship with Other Autonomously Replicating RNA Species

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and >4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5′ terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b -oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3′ termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Qβ and "variant" RNAs that serve as in vitro templates for the Qβ replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Structural consensus of RNA templates replicated by Qβ replicase

Qβ replicase replicates a variety of enzyme-specific small RNAs in addition to the phage genomic RNA. The sequence analysis has revealed that all these RNAs are potentially capable of forming a consensus secondary structure element. It represents a stalk which is formed by the 5′-GGG… and …CCCA-3′ complementary stretches at the termini of the replicating RNA molecules and adjacent 5′- and 3′-hairpins, which may form a stacking with the stalk. The structure found is rather similar to the analogous structure in the tRNA molecule. The genomic RNA of the Qβ phage and other related phages can also form a similar structural element.

Quantitative analysis of mutation and selection in self-replicating RNA

Mutation and selection as principles of Darwinian evolution have contributed a wealth to qualitative insight and understanding of complex biological organizations. However, for quantitative measurements of Darwinian evolution, only model systems are sufficiently simple to allow calculation of values for the relevant evolution parameters. The model system used for our study comprises short-chained RNA species whose self-replication is catalyzed by Qβ replicase. In this system, phenotypic expression of a genotype is reduced to its efficiency in directing its own synthesis.