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Tracey A. Lincoln and Gerald F. Joyce. Self-Sustained Replication of an RNA Enzyme // Science, Vol. 323. no. 5918, pp. 1229 - 1232 (27 February 2009).

Originally published in Science Express on 8 January 2009

Science 27 February 2009:

Vol. 323. no. 5918, pp. 1229 - 1232

DOI: 10.1126/science.1167856

Reports

Self-Sustained Replication of an RNA Enzyme

Tracey A. Lincoln and Gerald F. Joyce^*

An RNA enzyme that catalyzes the RNA-templated joining of RNAwas converted to a format whereby two enzymes catalyze eachother's synthesis from a total of four oligonucleotide substrates.These cross-replicating RNA enzymes undergo self-sustained exponentialamplification in the absence of proteins or other biologicalmaterials. Amplification occurs with a doubling time of about1 hour and can be continued indefinitely. Populations of variouscross-replicating enzymes were constructed and allowed to competefor a common pool of substrates, during which recombinant replicatorsarose and grew to dominate the population. These replicatingRNA enzymes can serve as an experimental model of a geneticsystem. Many such model systems could be constructed, allowingdifferent selective outcomes to be related to the underlyingproperties of the genetic system.

Department of Chemistry, Department of Molecular Biology, and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

^* To whom correspondence should be addressed. E-mail: gjoyce@scripps.edu

A long-standing research goal has been to devise a nonbiologicalsystem that undergoes replication in a self-sustained manner,brought about by enzymatic machinery that is part of the systembeing replicated. One way to realize this goal, inspired bythe notion of primitive RNA-based life, would be for an RNAenzyme to catalyze the replication of RNA molecules, includingthe RNA enzyme itself (1–4). This has now been achievedin a cross-catalytic system involving two RNA enzymes that catalyzeeach other's synthesis from a total of four component substrates.

The "R3C" RNA enzyme is an RNA ligase that binds two oligonucleotidesubstrates through Watson-Crick pairing and catalyzes nucleophilicattack of the 3'-hydroxyl of one substrate on the 5'-triphosphateof the other, forming a 3',5'-phosphodiester and releasing inorganicpyrophosphate (5). The R3C ligase was configured to self-replicateby joining two RNA molecules to produce another copy of itself(6). This process was inefficient because the substrates formeda nonproductive complex that limited the extent of exponentialgrowth, with a doubling time of about 17 hours and no more thantwo successive doublings.

The R3C ligase subsequently was converted to a cross-catalyticformat (Fig. 1A), whereby a plus-strand RNA enzyme (E) catalyzesthe joining of two substrates (A' and B') to form a minus-strandenzyme (E'), which in turn catalyzes the joining of two substrates(A and B) to form a new plus-strand enzyme (7, 8). This toowas inefficient because of the formation of nonproductive complexesand the slow underlying rate of the two enzymes. The enzymesE and E' operate with a rate constant of only

0.03 min^–1and a maximum extent of only 10 to 20% (9). These rates areabout 10 times slower than that of the parental R3C ligase (5),and when the two cross-catalytic reactions are carried out withina common mixture the rates are even slower (7).

Fig. 1. Cross-replicating RNA enzymes. (A) The enzyme E' (gray) catalyzes ligation of substrates A and B (black) to form the enzyme E, whereas E catalyzes ligation of A' and B' to form E'. The two enzymes dissociate to provide copies that can catalyze another reaction. (B) Sequence and secondary structure of the complex formed between the enzyme and its two substrates (E', A, and B are shown; E, A', and B' are the reciprocal). The curved arrow indicates the site of ligation. Solid boxes indicate critical wobble pairs that provide enhanced catalytic activity. Dashed boxes indicate paired regions and catalytic nucleotides that were altered to construct various cross replicators. (C) Variable portion of 12 different E enzymes. The corresponding E' enzymes have a complementary sequence in the paired region and the same sequence of catalytic nucleotides (alterations relative to the E1 enzyme are highlighted). [View Larger Version of this Image (24K GIF file)]

The catalytic properties of the cross-replicating RNA enzymeswere improved by the use of in vitro evolution, optimizing thetwo component reactions in parallel and seeking solutions thatwould apply to both reactions when conducted in the cross-catalyticformat (9). The 5'-triphosphate–bearing substrate wasjoined to the enzyme via a hairpin loop (B' to E and B to E'),and nucleotides within both the enzyme and the separate 3'-hydroxyl–bearingsubstrate (A' and A) were randomized at a frequency of 12% perposition. The two resulting populations of molecules were subjectedto six rounds of stringent in vitro selection, selecting fortheir ability to react in progressively shorter times, rangingfrom 2 hours to 10 ms. Mutagenic polymerase chain reaction wasperformed after the third round to maintain diversity in thepopulation. After the sixth round, individuals were cloned fromboth populations and sequenced. There was substantial sequencevariability among the clones, but all contained mutations justupstream from the ligation junction that resulted in a G·Uwobble pair at this position.

The G·U pair was installed in both enzymes and both 3'-hydroxyl–bearingsubstrates (Fig. 1B). In the trimolecular reaction (with twoseparate substrates), the optimized enzymes E and E' exhibiteda rate constant of 1.3 and 0.3 min^–1 with a maximum extentof 92 and 88%, respectively. The optimized enzymes underwentrobust exponential amplification at a constant temperature of42°C, with more than 25-fold amplification after 5 hours,followed by a leveling off as the supply of substrates becamedepleted (Fig. 2A). The data fit well to the logistic growthequation [E]_t = a/(1 + be^–ct), where [E]_t is the concentrationof E (or E') at time t, a is the maximum extent of growth, bis the degree of sigmoidicity, and c is the exponential growthrate. For the enzymes E and E', the exponential growth ratewas 0.92 and 1.05 hour^–1, respectively.

Fig. 2. Self-sustained amplification of cross-replicating RNA enzymes. (A) The yield of both E (black curve) and E' (gray curve) increased exponentially before leveling off as the supply of substrates became exhausted. (B) Amplification was sustained by performance of a serial transfer experiment, allowing approximately 25-fold amplification before transferring 4% 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. [View Larger Version of this Image (13K GIF file)]

Exponential growth can be continued indefinitely in a serialtransfer experiment in which a portion of a completed reactionmixture is transferred to a new reaction vessel that containsa fresh supply of substrates. Six successive reactions werecarried out in this fashion, each 5 hours in duration and transferring4% of the material from one reaction mixture to the next. Thefirst mixture contained 0.1 µM E and 0.1 µM E',but all subsequent mixtures contained only those enzymes thatwere carried over in the transfer. Exponential growth was maintainedthroughout 30 hours total of incubation, with an overall amplificationof greater than 10⁸-fold for each of the two enzymes (Fig. 2B).

It is possible to construct variants of the cross-replicatingRNA enzymes that differ in the regions of Watson-Crick pairingbetween the cross-catalytic partners without markedly affectingreplication efficiency. These regions are located at the 5'and 3' ends of the enzyme (Fig. 1B). Four nucleotide positionsat both the 5' and 3' ends were varied, adopting the rule thateach region contains one G·C and three A·U pairsso that there would be no substantial differences in base-pairingstability. Of the 32 possible pairs of complementary sequencesfor each region, 12 were chosen as a set of designated pairings(Fig. 1C). Each pairing was associated with a particular sequencewithin the catalytic core of both members of a cross-replicatingpair. Twelve pairs of cross-replicating enzymes were synthesized,as well as the 48 substrates (12 each of A, A', B, and B') necessaryto support their exponential amplification. Each replicatorwas individually tested and demonstrated varying levels of catalyticactivity and varying rates of exponential growth (fig. S1).The pair shown in Fig. 1B (now designated E1 and E1') had thefastest rate of exponential growth, achieving about 20-foldamplification after 5 hours. The various cross-replicating enzymesshown in Fig. 1C had the following rank order of replicationefficiency: E1, E10, E5, E4, E6, E3, E12, E7, E9, E8, E2, E11.The top five replicators all achieved more than 10-fold amplificationafter 5 hours, and all except E11 achieved at least fivefoldamplification after 5 hours.

A serial-transfer experiment was initiated with a 0.1 µMconcentration each of E1 to E4 and E1' to E4' and a 5.0 µMconcentration of each of the 16 corresponding substrates. Sixteensuccessive transfers were carried out over 70 hours, transferring5% of the material from one reaction mixture to the next (fig.S2A). Individuals were cloned from the population after thefinal reaction and sequenced. Among 25 clones (sequencing E'only), there was no dominant replicator (fig. S2B). E1', E2',E3', and E4' all were represented, as well as 17 clones thatwere the result of recombination between a particular A' substrateand one of the three B' substrates other than its original partner(or similarly for A and B). Recombination occurs when an enzymebinds and ligates a mismatched substrate. In principle, anyA could become joined to any B or B', and any A' could becomejoined to any B' or B, resulting in 64 possible enzymes. Theset of replicators were designed so that cognate substrateshave a binding advantage of several kilocalories per mole ascompared with noncognate substrates (fig. S2C), but once a mismatchedsubstrate is bound and ligated, it forms a recombinant enzymethat also can cross-replicate. Recombinants can give rise toother recombinants, as well as revert back to nonrecombinants.Based on relative binding affinities, there are expected tobe preferred pathways for mutation, primarily involving substitutionamong certain A' or among certain B components (fig. S2D).

A second serial transfer experiment was initiated with a 0.1µM concentration each of all 12 pairs of cross-replicatingenzymes and a 5.0 µM concentration of each of the 48 correspondingsubstrates. This mixture allowed 132 possible pairs of recombinantcross-replicating enzymes as well as the 12 pairs of nonrecombinantcross-replicators. Twenty successive reactions were carriedout over 100 hours, transferring 5% of the material from onereaction mixture to the next, and achieving an overall amplificationof greater than 10²⁵-fold (Fig. 3A). Of 100 clones isolatedafter the final reaction (sequencing 50 E and 50 E'), only 7were nonrecombinants (Fig. 3B). The distribution was highlynonuniform, with sparse representation of molecules containingcomponents A6 to A12 and B5 to B12 (and reciprocal componentsB6' to B12' and A5' to A12'). The most frequently representedcomponents were A5 and B3 (and reciprocal components B5' andA3'). The three most abundant recombinants were A5B2, A5B3,and A5B4 (and their cross-replication partners), which togetheraccounted for one third of all clones.

Fig. 3. 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. 1C), amplification was sustained for 20 successive rounds of

20-fold amplification and 20-fold dilution. The concentrations of all E (black) and E' (gray) molecules were measured after each incubation. (B) Graphical representation of 50 E and 50 E' clones (dark and light columns, respectively) that were sequenced after the last incubation. The A and B (or B' and A') components of the various enzymes are shown on the horizontal axes, with nonrecombinant enzymes indicated by shaded boxes along the diagonal. The number of clones containing each combination of components is shown on the vertical axis. (C) Comparative growth of E1 (circles) and A5B3 (squares) in the presence of either their cognate substrates alone (solid symbols) or all substrates that were present during serial transfer (open symbols). (D) Growth of A5B3 (black curve) and B5'A3' (gray curve) 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. [View Larger Version of this Image (38K GIF file)]

In the presence of their cognate substrates alone, E1 remainedthe most efficient replicator, but in the presence of all 48substrates, the most efficient replicator was A5B3 (Fig. 3C).When the A5B3 replicator was provided with a mixture of substratescorresponding to the components of the three most abundant recombinants,its exponential growth rate was the highest measured for anyreplicator (Fig. 3D). The fitness of a pair of cross-replicatingenzymes depends on several factors, including their intrinsiccatalytic activity, exponential growth rate with cognate substrates,ability to withstand inhibition by other substrates in the mixture,and net rate of production through mutation among the variouscross-replicators. The A5B3 recombinant and its cross-replicationpartner B5'A3' have different catalytic cores (Fig. 1C), andboth exhibit comparable activity, accounting for their well-balancedrate of production throughout the course of exponential amplification(Fig. 3D). The selective advantage of this cross-replicatorappears to derive from its relative resistance to inhibitionby other substrates in the mixture (Fig. 3C) and its abilityto capitalize on facile mutation among substrates B2, B3, andB4 and among substrates A2', A3', and A4' that comprise themost abundant recombinants (fig. S2D).

Populations of cross-replicating RNA enzymes can serve as asimplified experimental model of a genetic system with, at present,two genetic loci and 12 alleles per locus. It is likely, however,that the number of alleles could be increased by exploitingmore than four nucleotide positions at the 5' and 3' ends ofthe enzyme and by relaxing the rule that these nucleotides formone G·C and three A·U pairs. In order to supportmuch greater complexity, it will be necessary to constrain theset of substrates, for example, by using the population of newlyformed enzymes to generate a daughter population of substrates(9). An important challenge for an artificial RNA-based geneticsystem is to support a broad range of encoded functions, wellbeyond replication itself. Ultimately, the system should provideopen-ended opportunities for discovering novel function, somethingthat probably has not occurred on Earth since the time of theRNA world but presents an increasingly tangible research opportunity.

References and Notes

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4. L. E. Orgel, Crit. Rev. Biochem. Mol. Biol. 39, 99 (2004). [CrossRef] [Web of Science] [Medline]
5. J. Rogers, G. F. Joyce, RNA 7, 395 (2001).[Abstract]
6. N. Paul, G. F. Joyce, Proc. Natl. Acad. Sci. U.S.A. 99, 12733 (2002).[Abstract/Free Full Text]
7. D.-E. Kim, G. F. Joyce, Chem. Biol. 11, 1505 (2004). [CrossRef] [Web of Science] [Medline]
8. K.-S. Kim, S. Oh, S. S. Yea, M.-Y. Yoon, D.-E. Kim, FEBS Lett. 582, 2745 (2008). [CrossRef] [Web of Science] [Medline]
9. Materials and methods are available as supporting material on Science Online.
10. The authors thank L. Orgel for many stimulating discussions. This research was supported by grants from NASA (NNX07AJ23G) and NIH (R01GM065130) and by the Skaggs Institute for Chemical Biology.