Emergence of a fast-reacting ribozyme that is capable of undergoing continuous evolution Sarah B. Voytek and Gerald F. Joyce* Departments of Chemistry and Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 Contributed by Gerald F. Joyce, August 8, 2007 (sent for review July 9, 2007) It is possible to evolve RNA enzymes in a continuous manner by moter-containing oligonucleotide substrate and two proteins, re- employing a simple serial-transfer procedure. This method was verse transcriptase and RNA polymerase, to bring about the previously applied only to descendants of one unusually fast- selective amplification of reactive ribozymes (Fig. 1). The RNA reacting RNA enzyme with RNA ligase activity. The present study population expands exponentially and can be diluted serially to establishes a second continuously evolving RNA enzyme, also with allow sustained exponential growth. The serial transfers also pro- RNA ligase activity, but with a completely independent evolution- vide an opportunity to change the selection pressures applied to the ary origin. Critical to achieving the fast catalytic rate necessary for evolving population, for example, lowering the Mg2ϩ concentration continuous evolution, development of this enzyme entailed the (15), raising or lowering the pH (16), or introducing a ribozyme- addition and evolutionary maturation of a 35-nucleotide accessory cleaving DNA enzyme (17). domain and the application of highly stringent selection pressure, Continuous evolution is the most efficient and powerful method with reaction times as short as 15 ms. Once established, continuous for evolving functional molecules in vitro, but thus far has only been evolution was carried out for 80 successive transfers, maintaining applied to descendants of the class I ligase. It has been hypothesized the population against an overall dilution of 10207-fold. The re- that continuous evolution could be used to evolve other ligase sulting RNA enzymes exhibited Ϸ105-fold improvement in catalytic ribozymes and perhaps RNA molecules that catalyze other bond- efficiency, compared with the starting molecules, and became forming reactions (18). However, no other ligase ribozyme has a dependent on a structural feature of the substrate that previously catalytic rate as fast as that of the class I ligase. A fast catalytic rate conferred no selective advantage. This adaptation was eliminated is essential for continuous evolution because the ribozyme must by deleting the substrate feature and then carrying out 20 addi- react before it becomes reverse transcribed. Only if the ribozyme tional transfers of continuous evolution, which resulted in mole- reacts before it is reverse transcribed will it acquire a promoter cules with even greater catalytic activity. Now that two distinct sequence that allows the resulting cDNA to give rise to progeny species of continuously evolving enzymes have been established, ribozymes. it is possible to conduct molecular ecology experiments in which The goal of the present study was to develop a second ribozyme the two are made to compete for limited resources within a that can evolve in a continuous manner. This goal is a challenge for common environment. RNA catalysis and an opportunity to develop a distinct species of continuously evolving ribozymes that might be made to operate in in vitro evolution ͉ RNA catalysis ͉ RNA ligase ͉ selection ͉ serial transfer competition or cooperation with the class I ligase. The DSL ligase ribozyme, described previously by Inoue and colleagues (8), was arwinian evolution has been applied to populations of RNA chosen as the starting material for this effort. The DSL ligase was Dmolecules in the laboratory to generate RNA enzymes (ri- obtained by using a combination of rational design and in vitro bozymes) that catalyze a variety of reactions (1–3). These experi- selection. The framework for this ribozyme is a structural scaffold ments involve repeated rounds of selective amplification and mu- based on a naturally occurring RNA motif, to which was added a tagenesis to drive the evolving population toward desired catalytic duplex region for templated ligation and a potential catalytic behaviors. Of the many ribozymes that have been obtained by in domain of 30 random-sequence nucleotides. A large pool of these vitro evolution, six different classes catalyze the RNA-templated RNA molecules was subjected to 10 rounds of in vitro selection, joining of an oligonucleotide 3Ј-hydroxyl and an oligonucleotide resulting in a variety of ligase ribozymes. The most active catalyst 5Ј-triphosphate, forming a 3Ј,5Ј-phosphodiester and releasing in- identified is the cis-DSL-1S ribozyme, which contains 140 nucleo- Ϫ1 organic pyrophosphate (4–9). This reaction is similar to that carried tides and has a kcat of 0.066 min and a Km of 940 nM, measured out by protein polymerases that catalyze the synthesis of biological in the presence of 50 mM MgCl2 and 200 mM KCl at pH 7.5 and RNAs. 37°C (8, 19). All six of the known 3Ј,5Ј-ligase ribozymes achieve rate enhance- The catalytic activity of the DSL ribozyme is noteworthy for a ments of several orders of magnitude, compared with the uncata- Ϫ Ϫ ligase obtained after only 10 rounds of selective amplification and lyzed rate of templated RNA ligation, which is Ϸ10 7 min 1 (10). derived from only 30 random-sequence nucleotides. The small size Notably, only derivatives of the class I ligase, first described by and modular construction of the DSL ligase were regarded as Bartel, Szostak, and colleagues (4, 11), have been able to achieve a advantageous features for the development of a second continu- Ͼ Ϫ1 catalytic rate of 1min . Because of the superior catalytic rate of ously evolving ribozyme. In the present study, the DSL ribozyme this ribozyme, it has been chosen as the starting point for several was structurally modified to accommodate the promoter- subsequent in vitro evolution experiments. One series of studies led containing substrate needed for continuous evolution, and an to the development of a sequence-general, RNA-dependent RNA accessory domain of 35 random-sequence nucleotides was added to polymerase ribozyme that can add as many as 20 NTPs (12, 13). This effort involved several structural modifications of the class I ligase, including the addition of a large random-sequence accessory Author contributions: S.B.V. and G.F.J. designed research; S.B.V. performed research; S.B.V. domain, followed by 24 rounds of stepwise evolution and extensive and G.F.J. analyzed data; and S.B.V. and G.F.J. wrote the paper. screening of cloned individuals. The authors declare no conflict of interest. Another line of investigation beginning with the class I ligase *To whom correspondence should be addressed. E-mail: [email protected]. resulted in the establishment of a system for the continuous in vitro This article contains supporting information online at www.pnas.org/cgi/content/full/ evolution of ribozymes (14). This system operates at a constant 0707490104/DC1. temperature within a common reaction vessel. It employs a pro- © 2007 by The National Academy of Sciences of the USA 15288–15293 ͉ PNAS ͉ September 25, 2007 ͉ vol. 104 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707490104 Downloaded by guest on September 23, 2021 prom (–) prom (–) 5´ OH pp 5´ 5´ pppA p 3´ 5´ 3´ 3´ RNA polymerase NTPs reverse prom (–) transcriptase prom (–) p 3´ p 3´ 5´ 5´ prom (+) dNTPs Fig. 1. Scheme for continuous evolution of ligase ribozymes. RNA and DNA are depicted as solid and open lines, respectively. Ligation of a chimeric DNA–RNA substrate by the ribozyme results in attachment of the T7 promoter sequence (prom) to the 5Ј end of the ribozyme. Reverse transcription of reacted, but not unreacted, ribozymes results in the formation of a cDNA that serves as a template for transcription of multiple progeny RNAs. provide the opportunity for substantial improvement in catalytic eventually reaching 30 s. The ligated RNAs, representing catalyt- rate. A population of these modified ribozymes was subjected to ically competent molecules, were isolated by gel electrophoresis and vigorous stepwise evolution, with reaction times as short as 15 ms, then amplified to generate progeny RNAs to begin the next round to drive the improvement of catalytic function. This stringent of evolution. During the first three rounds, the reaction mixture selection pressure ultimately resulted in a population of DSL- contained only ribozymes, substrate, 25 mM MgCl2, and 50 mM derived ribozymes that were capable of undergoing continuous evolution. Once established, continuous evolution can be continued indefinitely, resulting in further improvement in the catalytic ac- A tivity of the ribozymes. This work demonstrates that continuous 5´- CACTAAT AG C G T C A C U A U U pppA G G G A A G evolution can be applied to ribozymes in addition to the class I OH A U UUUUUA G U G A U AA UCC C U U C A U A ligase, setting the stage for molecular ecology studies in which two U U A A A or more species evolve within a common environment. UUUUUUG C G U A G U C U C A A U C C U G G C A U G C G U C A G A U A G G A U C G A 3´- C U C C U C Results G A AA Stepwise Evolution. The DSL ribozyme was modified in several ways U U C G to render it compatible with the continuous evolution system (Fig. C G C G 2A). Continuous evolution requires the RNA-catalyzed ligation of UU U a chimeric DNA–RNA substrate that has the sequence of an RNA G A polymerase promoter. This substrate is more AU-rich at its 3Ј end, C G C G compared with the preferred substrate for the DSL ribozyme, U A A C making it necessary to extend the template region of the ribozyme C so that it could bind the substrate with sufficient affinity.