An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency ( k _cat / K _m ) of ≈10 ⁹ M ⁻¹ ⋅min ⁻¹ under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg ²⁺ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag / pol , env , vpr , tat , and nef mRNAs.

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.

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 genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA.

▪ Abstract 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.

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.