De novo synthesis and development of an RNA enzyme

Abstract

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...

Coordinated control of a designed trans‐acting ligase ribozyme by a loop–receptor interaction

“…The original cisDSL ribozyme was constructed by design using a well-defined self-folding RNA as a structural scaffold [8]. In this study, we constructed a series of trans-acting DSL ribozymes using a modular replacement approach.…”

“…1A-left) [8]. We were able to derive a trans-acting ribozyme from DSL by modular engineering because DSL consists of highly modular local structures [8].…”

“…Many artificial ribozymes with a wide variety of catalytic functions have been obtained by practicing in vitro selection, demonstrating that RNA can be a versatile functional molecule [2]. Several artificial RNA ligase ribozymes have been reported [3][4][5][6][7][8]. Such ribozymes have been considered to be components of a self-replicating system in the RNA world [9,10].…”

“…1A-left) [8]. We were able to derive a trans-acting ribozyme from DSL by modular engineering because DSL consists of highly modular local structures [8]. cisDSL-1S, which is the most active variant of the cisDSL ribozymes, was dissected into a substrate unit (consisting of the P1 region) and a catalytic unit (consisting of the P3 region) by replacement of the triple helical scaffold (THS) with a loop-receptor interaction module ( Fig.…”

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

“…A large pool of these RNA molecules was subjected to 10 rounds of in vitro selection, resulting in a variety of ligase ribozymes. The most active catalyst identified is the cis-DSL-1S ribozyme, which contains 140 nucleotides and has a k cat of 0.066 min Ϫ1 and a K m of 940 nM, measured in the presence of 50 mM MgCl 2 and 200 mM KCl at pH 7.5 and 37°C (8,19).The catalytic activity of the DSL ribozyme is noteworthy for a ligase obtained after only 10 rounds of selective amplification and derived from only 30 random-sequence nucleotides. The small size and modular construction of the DSL ligase were regarded as advantageous features for the development of a second continuously evolving ribozyme.…”