The effect of cytidine on the structure and function of an RNA ligase ribozyme

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It is possible to evolve RNA enzymes in a continuous manner by employing a simple serial-transfer procedure. This method was previously applied only to descendants of one unusually fastreacting RNA enzyme with RNA ligase activity. The present study establishes a second continuously evolving RNA enzyme, also with RNA ligase activity, but with a completely independent evolutionary origin. Critical to achieving the fast catalytic rate necessary for continuous evolution, development of this enzyme entailed the addition and evolutionary maturation of a 35-nucleotide accessory domain and the application of highly stringent selection pressure, with reaction times as short as 15 ms. Once established, continuous evolution was carried out for 80 successive transfers, maintaining the population against an overall dilution of 10 207 -fold. The resulting RNA enzymes exhibited Ϸ10 5 -fold improvement in catalytic efficiency, compared with the starting molecules, and became dependent on a structural feature of the substrate that previously conferred no selective advantage. This adaptation was eliminated by deleting the substrate feature and then carrying out 20 additional transfers of continuous evolution, which resulted in molecules with even greater catalytic activity. Now that two distinct species of continuously evolving enzymes have been established, it is possible to conduct molecular ecology experiments in which the two are made to compete for limited resources within a common environment.in vitro evolution ͉ RNA catalysis ͉ RNA ligase ͉ selection ͉ serial transfer D arwinian evolution has been applied to populations of RNA molecules in the laboratory to generate RNA enzymes (ribozymes) that catalyze a variety of reactions (1-3). These experiments involve repeated rounds of selective amplification and mutagenesis to drive the evolving population toward desired catalytic behaviors. Of the many ribozymes that have been obtained by in vitro evolution, six different classes catalyze the RNA-templated joining of an oligonucleotide 3Ј-hydroxyl and an oligonucleotide 5Ј-triphosphate, forming a 3Ј,5Ј-phosphodiester and releasing inorganic pyrophosphate (4-9). This reaction is similar to that carried out by protein polymerases that catalyze the synthesis of biological RNAs.All six of the known 3Ј,5Ј-ligase ribozymes achieve rate enhancements of several orders of magnitude, compared with the uncatalyzed rate of templated RNA ligation, which is Ϸ10 Ϫ7 min Ϫ1 (10). Notably, only derivatives of the class I ligase, first described by Bartel, Szostak, and colleagues (4, 11), have been able to achieve a catalytic rate of Ͼ1 min Ϫ1 . Because of the superior catalytic rate of this ribozyme, it has been chosen as the starting point for several subsequent in vitro evolution experiments. One series of studies led to the development of a sequence-general, RNA-dependent RNA 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 larg...

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confidence: 99%

Emergence of a fast-reacting ribozyme that is capable of undergoing continuous evolution

Voytek

Joyce

2007

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“…An added consideration was to optimize the construct so that the presence of the template molecule would greatly enhance the rate of ligation of the substrates compared with their ligation in the absence of the template. The R3C RNA ligase ribozyme was chosen for this study because of its simple secondary structure, reasonably fast ligation rate, and ability to function in an intermolecular reaction format (23).…”

mentioning

confidence: 99%

“…The R3C ribozyme is an RNA-dependent RNA ligase that catalyzes the formation of a 3Ј,5Ј-phosphodiester linkage between two RNA molecules, with a catalytic rate of 0.32 min Ϫ1 (23). The ribozyme, which was obtained by in vitro evolution, catalyzes attack of the 3Ј-hydroxyl of the substrate on the ␣-phosphate of the 5Ј-triphosphate of the ribozyme, with concomitant release of inorganic pyrophosphate.…”

mentioning

confidence: 99%

“…The overall structure of the ribozyme is centered about a three-way junction, whose arms are defined by the P2, P3, and P4 stems. The ribozyme has been engineered to function in an intermolecular reaction format, with one substrate containing the attacking 3Ј-hydroxyl and a second substrate containing the 5Ј-triphosphate (23). In the present study, the R3C ligase was restructured as a symmetrical dimer, such that the ligated product would be identical to the template (Fig.…”

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confidence: 99%

See 1 more Smart Citation

A self-replicating ligase ribozyme

Paul

Joyce²

2002

Self Cite

233

161

A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min ؊1 , whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 ؋ 10 ؊11 M⅐min ؊1 . Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication. In living systems, replicative processes transfer genetic information from template nucleic acid molecules to newly synthesized, complementary products. Several nonenzymatic templatedependent ligation systems have been devised to study the role of a template in binding and positioning complementary substrates for covalent bond formation (1-5). These have included simple self-replicating systems of the form A ϩ B 3 T, where A and B are substrates that bind to a complementary template, T, and become joined to form a product molecule that is identical to the template (6-9). The unique aspect of self-replicating systems is that the reaction product has the potential to direct additional reactions. The system is termed autocatalytic when the product is an efficient template, and each covalent bond that is formed generates additional template molecules that can direct further joining reactions. The realization of autocatalytic behavior in a self-replicating system implies that sustained exponential growth may be possible.The self-replicating systems that have been studied to date use template molecules composed of nucleic acids (6, 7, 10), peptides (11-14), or small organic compounds (15-17). The nucleic acid-based systems are the most straightforward and rely on simple Watson-Crick pairing interactions between a short oligonucleotide template and two complementary oligonucleotide substrates (6, 7, 10). The substrates are bound at adjacent positions along the template and are joined through a reaction involving chemical groups at their opposed ends. Peptide-based self-replicating systems are similar, except that the components are oligopeptides that have the capacity to form ␣-helices. The template is the hydrophob...

“…7, which is published as supporting information on the PNAS web site)]. In contrast, the previously reported ligases with the exception of class I can use only limited combinations of the two substrate nucleotides at the reaction site (14,(24)(25)(26).…”

Section: ϫ3mentioning

confidence: 99%

De novo synthesis and development of an RNA enzyme

Ikawa

Tsuda²,

Matsumura³

et al. 2004

117

Arbitrary manipulation of molecular recognition at the atomic level has many applications. However, systematic design and de novo synthesis of an artificial enzyme based on such manipulation has been a long-standing challenge in the field of chemistry and biotechnology. In this report, we developed an artificial RNA ligase by implementing a synthetic strategy that fuses a series of 3D molecular modelings based on naturally occurring RNA-RNA recognition motifs with a small-scale combinatorial synthesis of a modular catalytic unit. The resulting ligase produces a 3-5 linkage in a template-directed manner for any combinations of two nucleotides at the reaction site. The reaction rate is 10 6 -fold over that of the uncatalyzed reaction with a yield higher than those of previously reported ligase ribozymes. The strategy may be applicable to the synthesis and development of a variety of nonnatural functional RNAs with defined 3D structures. Many RNA receptors and enzymes have been selected in vitro from combinatorial libraries that consist of a long random sequence (1). Anatomies of the selected RNAs are difficult to predict because their higher-order structures were not specified before the selection. However, if a precisely designed assemblage of well established modular units is used as a scaffold for preparing such functional RNAs, redesign of the resulting selected RNAs will be facilitated. They will thus enable a variety of artificial evolutionary pathways, starting from the selected molecule.In general, molecular design of biopolymers such as RNAs or proteins is difficult at the 3D level because of their highly complicated folding process. In the 1990s, biochemical and structural analyses revealed that many functional noncoding natural RNAs are organized into modules and fold into defined 3D structures (2-5). Moreover, several commonly used RNA-RNA binding motifs in these RNAs were identified by phylogenetic comparison (6) and high-resolution structural analyses (7-10). Consequently, it has become possible to design self-folding RNAs precisely by employing such motifs and mimicking the modular organization of natural RNAs (11-13). As one such example, we have previously reported the design of a self-folding RNA consisting of standard doublestranded helices connected by the two motifs: a tetraloopreceptor interaction and consecutive base-triples ( Fig. 1 A and B) (13). Results indicated that the constructed RNA folds compactly into the designed 3D structure.In this paper, we report the synthesis and development of an artificial RNA ligase as shown in the scheme (Fig. 1 A). First, a reaction site for RNA-RNA ligation was installed into the designed RNA scaffold, and a different region of the RNA was subject in vitro selection from a small combinatorial library to provide a catalytic center. The ligation reaction was chosen as a convenient target because several RNA ligases had already been obtained from large-scale pools by in vitro selection (ref. 14 and references cited therein). Biochemical characterization a...