Self-sustained replication of an RNA enzyme

Abstract

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.

Fig. 1

Scheme for cross-catalytic replication of RNA enzymes. (A) The enzyme E′ (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, while E catalyzes ligation of A′ and B′ to form E′. The two enzymes dissociate to provide copies of both E and E′ that each can catalyze another reaction. (B) Sequence and secondary structure of the complex formed between the cross-replicating RNA enzyme and its two substrates (E′, A, and B are shown; E, A′, and B′ are the reciprocal). Curved arrow indicates the site of ligation. Boxed residues indicate the sites of critical wobble pairs that provide enhanced catalytic activity compared to the parental R3C ligase.

Fig. 2

Self-sustained amplification of cross-replicating RNA enzymes. (A) The yield of both E (black) and E′ (gray) increased exponentially before leveling off as the supply of substrates became exhausted. (B) Amplification was sustained by performing a serial transfer experiment, allowing ~25-fold amplification before transferring 1/25th of the mixture to a new reaction vessel that contained a fresh supply of substrates. The concentrations of E and E′ were measured at the end of each incubation.

Fig. 3

Twelve pairs of cross-replicating RNA enzymes were constructed. Four nucleotides at the 5′ and 3′ ends of the enzyme were chosen as the sites for genotypic variation, and 11 nucleotides within the catalytic core were chosen as the corresponding sites for phenotypic variation (boxed regions). The sequence of these regions for each of the 12 E enzymes is shown at the right. The corresponding E′ enzymes have a complementary sequence in the genotype region and the same sequence in the catalytic core. Alterations of the catalytic core relative to the E1 enzyme are highlighted by black circles.

Fig. 4

Self-sustained amplification of a population of cross-replicating RNA enzymes, resulting in selection of the fittest replicators. (A) Beginning with 12 pairs of cross-replicating RNA enzymes (Fig. 3), amplification was sustained by serial transfer for 20 successive rounds of ~20-fold amplification and 20-fold dilution. The concentrations of all E (black) and E′ (gray) molecules were measured at the end of each incubation. (B) Graphical representation of the observed genotypes among 50 E and 50 E′ clones (dark and light columns, respectively) that were sequenced following the last incubation. The A and B (or B′ and A′) components of the various enzymes are shown on the horizontal axes, with non-recombinant enzymes indicated by shaded boxes along the diagonal. The number of clones containing each combination of components is shown on the vertical axis.

Fig. 5

Exponential amplification of the starting cross-replicating enzymes (E1 and E1′) and of the most efficient cross-replicator (A5B3 and B5′A3′) that emerged during the serial transfer experiment involving all 48 substrates (Fig. 4). (A) Comparative growth of E1 (circles) and A5B3 (squares) in the presence of either their cognate substrates alone (filled symbols) or all substrates that were present during serial transfer (open symbols). (B) Growth of A5B3 (black) and B5′A3′ (gray) in the presence of the eight substrates (A5, B2, B3, B4, B5′, A2′, A3′, and A4′) that comprise the three most abundant cross-replicating enzymes.