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.

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.

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.

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.

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.

Ligand-dependent exponential amplification of a self-replicating L-RNA enzyme.

A nuclease-resistant RNA enzyme, constructed entirely from L-ribonucleotides, was shown to undergo ligand-dependent, self-sustained replication with exponential growth. The catalytic motif is based on a previously described RNA ligase that can undergo either self- or cross-replication but had been limited in its application to ligand sensing due to its susceptibility to degradation by ribonucleases. The self-replicating RNA enzyme and its RNA substrates were prepared synthetically from either D- or L-nucleoside phosphoramidites. The D and L reaction systems undergo isothermal, ligand-dependent exponential amplification in the same manner, but only the l system is impervious to ribonucleases and can operate, for example, in the presence of human serum. This system has potential for the quantitative detection of various ligands that are present within biological or environmental samples. In addition, this work provides the first demonstration of the self-sustained exponential amplification of nonbiological molecules.

Chemical Triphosphorylation of Oligonucleotides.

The 5'-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5'-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible. This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

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.

Conformational analysis of DNA repair intermediates by time-resolved fluorescence spectroscopy.

DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NMR and X-ray crystallography. Here, we used time-resolved fluorescence spectroscopy to examine the conformational dynamics of linear, nicked, gapped, and bulged DNA in the absence of protein enzymes. This analysis reveals that damaged DNA is polymorphic in nature and able to adopt multiple individual conformations. We show that DNA repair intermediates that contain a one-nucleotide gap and bulge have a significant propensity to adopt conformations in which the orphan base resides outside the DNA helix, while DNA structures damaged by a nick or two-nucleotide gap favor intrahelical conformations. Because changes in DNA conformation appear to guide the recognition of DNA repair enzymes, we suggest that the current approach could be used to study the mechanism of DNA repair.

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.