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.
Self-Sustained Replication of an RNA Enzyme Lincoln, Tracey A; Joyce, Gerald F
Science (American Association for the Advancement of Science),
02/2009, Letnik:
323, Številka:
5918
Journal Article
Recenzirano
Odprti dostop
An RNA enzyme that catalyzes the RNA-templated joining of RNA was converted to a format whereby two enzymes catalyze each other's synthesis from a total of four oligonucleotide substrates. These ...cross-replicating RNA enzymes undergo self-sustained exponential amplification in the absence of proteins or other biological materials. Amplification occurs with a doubling time of about 1 hour and can be continued indefinitely. Populations of various cross-replicating enzymes were constructed and allowed to compete for a common pool of substrates, during which recombinant replicators arose and grew to dominate the population. These replicating RNA enzymes can serve as an experimental model of a genetic system. Many such model systems could be constructed, allowing different selective outcomes to be related to the underlying properties of the genetic system.
Forty Years of In Vitro Evolution Joyce, Gerald F
Angewandte Chemie (International ed.),
01/2007, Letnik:
46, Številka:
34
Journal Article
Recenzirano
It has been 40 years since Spiegelman and co-workers demonstrated how RNA molecules can be evolved in the test tube. This result established Darwinian evolution as a chemical process and paved the ...way for the many directed evolution experiments that followed. Chemists can benefit from reflecting on Spiegelman's studies and the subsequent advances, which have taken the field to the brink of the generation of life itself in the laboratory. This Review summarizes the concepts and methods for the directed evolution of RNA molecules in vitro.
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.
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.
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(4H)-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.
An l-RNA aptamer was developed that binds the natural d-form of the HIV-1 trans-activation responsive (TAR) RNA. The aptamer initially was obtained as a d-aptamer against l-TAR RNA through in vitro ...selection. Then the corresponding l-aptamer was prepared by chemical synthesis and used to bind the desired target. The l-aptamer binds d-TAR RNA with a K d of 100 nM. It binds d-TAR exclusively at the six-nucleotide distal loop, but does so through tertiary interactions rather than simple Watson–Crick pairing. This complex is the first example of two nucleic acids molecules of opposing chirality that interact through a mode of binding other than primary structure. Binding of the l-aptamer to d-TAR RNA inhibits formation of the Tat-TAR ribonucleoprotein complex that is essential for TAR function. This suggests that l-aptamers, which are intrinsically resistant to degradation by ribonucleases, might be pursued as an alternative to antisense oligonucleotides to target structured RNAs of biological or therapeutic interest.
Just as Darwinian evolution in nature has led to the development of many
sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful
approach for obtaining similar results in the ...laboratory. This review focuses
on the development of nucleic acid enzymes starting from a population of
random-sequence RNA or DNA molecules. In order to illustrate the principles and
practice of in vitro evolution, two especially well-studied categories of
catalytic nucleic acid are considered: RNA enzymes that catalyze the
template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of
RNA. The former reaction, which involves attack of a 2′- or
3′-hydroxyl on the α-phosphate of a 5′-triphosphate, is more
difficult. It requires a comparatively larger catalytic motif, containing more
nucleotides than can be sampled exhaustively within a starting population of
random-sequence RNAs. The latter reaction involves deprotonation of the
2′-hydroxyl adjacent to the cleavage site, resulting in cleaved products
that bear a 2′,3′-cyclic phosphate and 5′-hydroxyl. The
difficulty of this reaction, and therefore the complexity of the corresponding
DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal
cation or small molecule, is present in the reaction mixture.
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.
All known examples of life belong to the same biology, but there is increasing enthusiasm among astronomers, astrobiologists, and synthetic biologists that other forms of life may soon be discovered ...or synthesized. This enthusiasm should be tempered by the fact that the probability for life to originate is not known. As a guiding principle in parsing potential examples of alternative life, one should ask: How many heritable "bits" of information are involved, and where did they come from? A genetic system that contains more bits than the number that were required to initiate its operation might reasonably be considered a new form of life.
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