RNA-catalyzed evolution of catalytic RNA.

An RNA polymerase ribozyme that was obtained by directed evolution can propagate a functional RNA through repeated rounds of replication and selection, thereby enabling Darwinian evolution. Earlier versions of the polymerase did not have sufficient copying fidelity to propagate functional information, but a new variant with improved fidelity can replicate the hammerhead ribozyme through reciprocal synthesis of both the hammerhead and its complement, with the products then being selected for RNA-cleavage activity. Two evolutionary lineages were carried out in parallel, using either the prior low-fidelity or the newer high-fidelity polymerase. The former lineage quickly lost hammerhead functionality as the population diverged toward random sequences, whereas the latter evolved new hammerhead variants with improved fitness compared to the starting RNA. The increase in fitness was attributable to specific mutations that improved the replicability of the hammerhead, counterbalanced by a small decrease in hammerhead activity. Deep sequencing analysis was used to follow the course of evolution, revealing the emergence of a succession of variants that progressively diverged from the starting hammerhead as fitness increased. This study demonstrates the critical importance of replication fidelity for maintaining heritable information in an RNA-based evolving system, such as is thought to have existed during the early history of life on Earth. Attempts to recreate RNA-based life in the laboratory must achieve further improvements in replication fidelity to enable the fully autonomous Darwinian evolution of RNA enzymes as complex as the polymerase itself.

RNA Polymerase Ribozyme That Recognizes the Template-Primer Complex through Tertiary Interactions.

RNA enzymes (ribozymes) often rely on specific base-pairing interactions to engage RNA substrates, which limits the substrate sequence generality of these enzymes. An RNA polymerase ribozyme that was previously optimized by directed evolution to operate in a more efficient and sequence-general manner can now recognize the RNA template, RNA primer, and incoming nucleoside 5'-triphosphate (NTP) entirely through tertiary interactions. As with proteinaceous polymerases, these tertiary interactions are largely agnostic to the sequence of the template, which is an essential property for the unconstrained transmission of genetic information. The polymerase ribozyme exhibits Michaelis-Menten saturation kinetics, with a catalytic rate of 0.1-1 min-1 and a Km of 0.1-1 µM. Earlier forms of the polymerase did not exhibit a saturable substrate binding site, but this property emerged over the course of directed evolution as the ribozyme underwent a structural rearrangement of its catalytic center. The optimized polymerase makes tertiary contacts with both the template and primer, including a critical interaction at the C2' position of the template nucleotide that opposes the 3'-terminal nucleotide of the primer. UV cross-linking studies paint a picture of how several portions of the ribozyme, including regions that were remodeled by directed evolution, come together to position the template, primer, and NTP within the active site for RNA polymerization.

Discovery of small molecules that target a tertiary-structured RNA.

There is growing interest in therapeutic intervention that targets disease-relevant RNAs using small molecules. While there have been some successes in RNA-targeted small-molecule discovery, a deeper understanding of structure-activity relationships in pursuing these targets has remained elusive. One of the best-studied tertiary-structured RNAs is the theophylline aptamer, which binds theophylline with high affinity and selectivity. Although not a drug target, this aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore, no compound has been shown to bind the theophylline aptamer with greater affinity than theophylline itself. However, by carrying out a high-throughput screen of low-molecular-weight compounds, several unique hits were identified that are chemically distinct from theophylline and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures were determined to investigate the binding mode of theophylline and four of the best hits. These structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity for other ligands to bind more tightly in this pocket by forming additional hydrogen-bonding interactions. These results give encouragement that the same approaches to drug discovery that have been applied so successfully to proteins can also be applied to RNAs.

Cross-Chiral, RNA-Catalyzed Exponential Amplification of RNA.

Informational macromolecules in biology are composed of subunits of a single handedness, d-nucleotides in nucleic acids and l-amino acids in proteins. Although this chiral uniformity may be expedient, it is not a chemical necessity, as demonstrated by the recent example of an RNA enzyme that catalyzes the RNA-templated polymerization of RNA molecules of the opposite handedness. This reaction, when carried out iteratively, can provide the basis for exponential amplification of RNA molecules and the information they contain. By carrying out thermal cycling, analogous to the polymerase chain reaction, and supplying oligonucleotide building blocks that comprise both the functional strand of RNA and its complement, cross-chiral exponential amplification was achieved. This process was used to amplify the l-RNA form of the hammerhead ribozyme, catalyzed by the d-RNA form of the polymerase. The resulting l-hammerhead exhibits the expected activity in cleaving a corresponding l-RNA substrate. Exponential amplification was also carried out within individual droplets of a water-in-oil emulsion. The ability to amplify enantio-RNAs, both in bulk solution and within compartments, provides a means to evolve cross-chiral RNA polymerases based on the function of the RNAs they produce.

Witnessing the structural evolution of an RNA enzyme.

An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, raising the possibility that further improvement of polymerase activity may be achievable.

Kinetic Effects of ß,γ-Modified Deoxynucleoside 5'-Triphosphate Analogues on RNA-Catalyzed Polymerization of DNA.

A recently described DNA polymerase ribozyme, obtained by in vitro evolution, provides the opportunity to investigate mechanistic features of RNA catalysis using methods that previously had only been applied to DNA polymerase proteins. Insight can be gained into the transition state of the DNA polymerization reaction by studying the behavior of various ß,γ-bridging substituted methylene (CXY; X, Y = H, halo, methyl) or imido (NH) dNTP analogues that differ with regard to the pKa4 of the bisphosphonate or imidodiphosphate leaving group. The apparent rate constant (kpol) of the polymerase ribozyme was determined for analogues of dGTP and dCTP that span a broad range of acidities for the leaving group, ranging from 7.8 for the CF2-bisphosphonate to 11.6 for the CHCH3-bisphosphonate. A Brønsted plot of log(kpol) versus pKa4 of the leaving group demonstrates linear free energy relationships (LFERs) for dihalo-, monohalo-, and non-halogen-substituted analogues of the dNTPs, with negative slopes, as has been observed for DNA polymerase proteins. The unsubstituted dNTPs have a faster catalytic rate than would be predicted from consideration of the linear free energy relationship alone, presumably due to a relatively more favorable interaction of the ß,γ-bridging oxygen within the active site. Although the DNA polymerase ribozyme is considerably slower than DNA polymerase proteins, it exhibits a similar LFER fingerprint, suggesting mechanistic commonality pertaining to the buildup of negative charge in the transition state, despite the very different chemical compositions of the two catalysts.

RNA-Catalyzed Cross-Chiral Polymerization of RNA.

Biology relies almost exclusively on homochiral building blocks to drive the processes of life. Yet cross-chiral interactions can occur between macromolecules of the opposite handedness, including a previously described polymerase ribozyme that catalyzes the template-directed synthesis of enantio-RNA. The present study sought to optimize and generalize this activity, employing in vitro evolution to select cross-chiral polymerases that use either mono- or trinucleotide substrates that are activated as the 5'-triphosphate. There was only modest improvement of the former activity, but dramatic improvement of the latter, which enables the trinucleotide polymerase to react 102-103-fold faster than its ancestor and to accept substrates with all possible sequence combinations. The evolved ribozyme can assemble long RNAs from a mixture of trinucleotide building blocks, including a two-fragment form of the ancestral polymerase ribozyme. Further improvement of this activity could enable the generalized cross-chiral replication of RNA, which would establish a new paradigm for the chemical basis of Darwinian evolution.

Thermal Habitat for RNA Amplification and Accumulation.

The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.

An RNA polymerase ribozyme that synthesizes its own ancestor.

The RNA-based organisms from which modern life is thought to have descended would have depended on an RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Such a polymerase must have been capable of copying structured RNAs with high efficiency and high fidelity to maintain genetic information across successive generations. Here the class I RNA polymerase ribozyme was evolved in vitro for the ability to synthesize functional ribozymes, resulting in the markedly improved ability to synthesize complex RNAs using nucleoside 5'-triphosphate (NTP) substrates. The polymerase is descended from the class I ligase, which contains the same catalytic core as the polymerase. The class I ligase can be synthesized by the improved polymerase as three separate RNA strands that assemble to form a functional ligase. The polymerase also can synthesize the complement of each of these three strands. Despite this remarkable level of activity, only a very small fraction of the assembled ligases retain catalytic activity due to the presence of disabling mutations. Thus, the fidelity of RNA polymerization should be considered a major impediment to the construction of a self-sustained, RNA-based evolving system. The propagation of heritable information requires both efficient and accurate synthesis of genetic molecules, a requirement relevant to both laboratory systems and the early history of life on Earth.

RNA-Catalyzed Polymerization of Deoxyribose, Threose, and Arabinose Nucleic Acids.

An RNA-dependent RNA polymerase ribozyme that was highly optimized through in vitro evolution for the ability to copy a broad range of template sequences exhibits promiscuity toward other nucleic acids and nucleic acid analogues, including DNA, threose nucleic acid (TNA), and arabinose nucleic acid (ANA). By operating on various RNA templates, the ribozyme catalyzes multiple successive additions of DNA, TNA, or ANA monomers, although with reduced efficiency compared to RNA monomers. The ribozyme can also copy DNA or TNA templates to complementary RNAs, and to a lesser extent it can operate when both the template and product strands are composed of DNA, TNA, or ANA. These results suggest that polymerase ribozymes, which are thought to have replicated RNA genomes during the early history of life, could have transferred RNA-based genetic information to and from DNA, enabling the emergence of DNA genomes prior to the emergence of proteins. In addition, genetic systems based on nucleic acid-like molecules, which have been proposed as precursors or contemporaries of RNA-based life, could have been operated upon by a promiscuous polymerase ribozyme, thus enabling the evolutionary transition between early genetic systems.

Mapping a Systematic Ribozyme Fitness Landscape Reveals a Frustrated Evolutionary Network for Self-Aminoacylating RNA.

Molecular evolution can be conceptualized as a walk over a "fitness landscape", or the function of fitness (e.g., catalytic activity) over the space of all possible sequences. Understanding evolution requires knowing the structure of the fitness landscape and identifying the viable evolutionary pathways through the landscape. However, the fitness landscape for any catalytic biomolecule is largely unknown. The evolution of catalytic RNA is of special interest because RNA is believed to have been foundational to early life. In particular, an essential activity leading to the genetic code would be the reaction of ribozymes with activated amino acids, such as 5(4 H)-oxazolones, to form aminoacyl-RNA. Here we combine in vitro selection with a massively parallel kinetic assay to map a fitness landscape for self-aminoacylating RNA, with nearly complete coverage of sequence space in a central 21-nucleotide region. The method (SCAPE: sequencing to measure catalytic activity paired with in vitro evolution) shows that the landscape contains three major ribozyme families (landscape peaks). An analysis of evolutionary pathways shows that, while local optimization within a ribozyme family would be possible, optimization of activity over the entire landscape would be frustrated by large valleys of low activity. The sequence motifs associated with each peak represent different solutions to the problem of catalysis, so the inability to traverse the landscape globally corresponds to an inability to restructure the ribozyme without losing activity. The frustrated nature of the evolutionary network suggests that chance emergence of a ribozyme motif would be more important than optimization by natural selection.

Protocells and RNA Self-Replication.

The general notion of an "RNA world" is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA, and RNA molecules were the chief agents of catalytic function. Assuming that all of the components of RNA were available in some prebiotic locale, these components could have assembled into activated nucleotides that condensed to form RNA polymers, setting the stage for the chemical replication of polynucleotides through RNA-templated RNA polymerization. If a sufficient diversity of RNAs could be copied with reasonable rate and fidelity, then Darwinian evolution would begin with RNAs that facilitated their own reproduction enjoying a selective advantage. The concept of a "protocell" refers to a compartment where replication of the primitive genetic material took place and where primitive catalysts gave rise to products that accumulated locally for the benefit of the replicating cellular entity. Replication of both the protocell and its encapsulated genetic material would have enabled natural selection to operate based on the differential fitness of competing cellular entities, ultimately giving rise to modern cellular life.

3'-End labeling of nucleic acids by a polymerase ribozyme.

A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.

A reverse transcriptase ribozyme.

A highly evolved RNA polymerase ribozyme was found to also be capable of functioning as a reverse transcriptase, an activity that has never been demonstrated before for RNA. This activity is thought to have been crucial for the transition from RNA to DNA genomes during the early history of life on Earth, when it similarly could have arisen as a secondary function of an RNA-dependent RNA polymerase. The reverse transcriptase ribozyme can incorporate all four dNTPs and can generate products containing up to 32 deoxynucleotides. It is likely that this activity could be improved through evolution, ultimately enabling the synthesis of complete DNA genomes. DNA is much more stable compared to RNA and thus provides a larger and more secure repository for genetic information.

Real-Time Detection of a Self-Replicating RNA Enzyme.

A system was developed to detect the self-replication of an RNA enzyme in real time. The enzyme is an RNA ligase that undergoes exponential amplification at a constant temperature and can be made to operate in a ligand-dependent manner. The real-time system is based on a fluorimetric readout that directly couples the ligation event to an increase in florescence signal that can be monitored using standard instrumentation. The real-time system can also operate entirely with l-RNA, which is not susceptible to degradation by ribonucleases that are present in biological samples. The system is analogous to real-time PCR, but with the potential to detect small molecules, proteins, and other targets that can be recognized by a suitable aptamer. The ligand-dependent self-replication of RNA has potential applications in molecular diagnostics and biosensing that benefit from the rapid, precise, and real-time detection of various target molecules.

Amplification of RNA by an RNA polymerase ribozyme.

In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins.

Specific Inhibition of MicroRNA Processing Using L-RNA Aptamers.

In vitro selection was used to obtain l-RNA aptamers that bind the distal stem-loop of various precursor microRNAs (pre-miRs). These l-aptamers, termed "aptamiRs", bind their corresponding pre-miR target through highly specific tertiary interactions rather than Watson-Crick pairing. Formation of a pre-miR-aptamiR complex inhibits Dicer-mediated processing of the pre-miR, which is required to form the mature functional microRNA. One of the aptamiRs, which was selected to bind oncogenic pre-miR-155, inhibits Dicer processing under simulated physiological conditions, with an IC50 of 87 nM. Given that l-RNAs are intrinsically resistant to nuclease degradation, these results suggest that aptamiRs might be pursued as a new class of miR inhibitors.

An L-RNA Aptamer that Binds and Inhibits RNase.

L-RNA aptamers were developed that bind to barnase RNase and thereby inhibit the function of the enzyme. These aptamers were obtained by first carrying out in vitro selection of D-RNAs that bind to the full-length synthetic D-enantiomer of barnase, then reversing the mirror and preparing L-RNAs of identical sequence that similarly bind to natural L-barnase. The resulting L-aptamers bind L-barnase with an affinity of ∼100 nM and function as competitive inhibitors of enzyme cleavage of D-RNA substrates. L-RNA aptamers are resistant to degradation by ribonucleases, thus enabling them to function in biological samples, most notably for applications in molecular diagnostics and therapeutics. In addition to the irony of using RNA to inhibit RNase, L-RNA aptamers such as those described here could be used to measure the concentration or inhibit the function of RNase in the laboratory or in biological systems.