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Yoshiya Ikawa

Publications and source records attributed to Yoshiya Ikawa.

At least 19 recordsLinked to original sources
PubMed2006

Construction of an artificial ribozyme which ligates an RNA fragment activated by nicotinamide mononucleotide.

A new strategy termed "Design & Selection" has been developed for construction of artificial ribozymes. In this strategy, two distinct approaches (de novo rational design and in vitro selection) were successfully combined. De novo rational design was employed for construction of a structural scaffold of a new ribozyme whereas in vitro selection was adopted for isolation of a catalytic unit, sequence and structure of which were unpredictable. Using this strategy, a ligase ribozyme (DSL ribozyme) has been isolated. To validate versatility of Design & Selection strategy, we attempted to isolate a new catalytic unit utilizing a nicotinamide mononucleotide as a leaving group in RNA-RNA ligation reaction. Into the de novo designed scaffold RNA, 45 random nucleotides were inserted. The resulting RNA library consisting of the scaffold and randomized regions was subjected to in vitro selection. Using the library with 10(14) sequence diversity, a new class of ribozyme bearing a novel catalytic unit sequence was successfully isolated.

Catalytic Domain↗
PubMed2005

Structural and biochemical characterization of DSL ribozyme.

We recently reported on the molecular design and synthesis of a new RNA ligase ribozyme (DSL), whose active site was selected from a sequence library consisting of 30 random nucleotides set on a defined 3D structure of a designed RNA scaffold. In this study, we report on the structural and biochemical analyses of DSL. Structural analysis indicates that the active site, which consists of the selected sequence, attaches to the folded scaffold as designed. To see whether DSL resembles known ribozymes, a biochemical assay was performed. Metal-dependent kinetic studies suggest that the ligase requires Mg2+ ions. The replacement of Mg2+ with Co(NH3)6(3+) prohibits the reaction, indicating that DSL requires innersphere coordination of Mg2+ for a ligation reaction. The results show that DSL has requirements similar to those of previously reported catalytic RNAs.

Base Sequence↗
PubMed2005

Redesign of an artificial ligase ribozyme based on the analysis of its structural elements.

The catalytic and folding properties of "DSL ribozyme" were investigated. This artificial ligase ribozyme was constructed by installing a catalytic unit to a designed self-folding RNA. The self-folding RNA was composed of three helices connected via two tertiary interactions that served as scaffolding in the molecular design. The present analysis revealed that the tertiary interaction between the GAAA loop and its specific receptor plays a crucial role in the folding of the active structure and the precise positioning of the catalytic site. On the basis of the analyses, the ribozyme was redesigned and converted to two advanced forms--a smaller derivative with appreciable catalytic activity and a derivative with RNA polymerase-like activity. The study demonstrates that redesign of an artificial ribozyme is effective and efficient if its structural elements are finely resolved. This kind of molecular transformation should serve as a prototypic model for understanding the molecular organization and evolution of naturally occurring ribozymes.

Catalytic Domain↗
PubMed2005

Rational installation of an allosteric effector on a designed ribozyme.

We designed and constructed an allosterically controllable ribozyme with rational molecular design. An allosteric modular unit that is under the control of an organic molecule was installed to an artificially designed ribozyme on the basis of the fact that the RNA is an assemblage of functional modular units. The ribozyme was successfully converted to allosterically controllable form.

Allosteric Regulation↗
PubMed2004

Generation of a catalytic module on a self-folding RNA.

It is theoretically possible to obtain a catalytic site of an artificial ribozyme from a random sequence consisting of a limited numbers of nucleotides. However, this strategy has been inadequately explored. Here, we report an in vitro selection technique that exploits modular construction of a structurally constrained RNA to acquire a catalytic site for RNA ligation from a short random sequence. To practice the selection, a sequence of 30 nucleotides was located close to the putative reaction site in a derivative of a naturally occurring self-folding RNA whose crystal structure is known. RNAs whose activity depended on the starting three-dimensional structure were selected with 3'-5' ligation specificity, indicating that the strategy can be used to acquire a variety of catalytic sites and other functional RNA modules.

Animals↗
PubMed2004

De novo synthesis and development of an RNA enzyme.

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.

Base Sequence↗
PubMed2004

A ligase ribozyme obtained from a structured pool.

Here we report the in vitro selection experiment employed to construct a catalytic site on a self-folding RNA. To practice the selection, the sequence of 30 nucleotides was located in close proximity of the putative reaction site in a derivative of a naturally occurring RNA with a well known 3D structure. The acquired catalytic RNA retained the starting 3D structure and was successfully reconstructed according to its modular structure.

Animals↗
PubMed2003

Artificial modules for enhancing rate constants of a Group I intron ribozyme without a P4-P6 core element.

In this paper we report newly selected artificial modules that enhance the kcat values comparable with or higher than those of the wild-type ribozyme with broad substrate specificity. The elements required for the catalysis of Group I intron ribozymes are concentrated in the P3-P7 domain of their core region, which consists of two conserved helical domains, P4-P6 and P3-P7. Previously, we reported the in vitro selection of artificial modules residing at the peripheral region of a mutant Group I ribozyme lacking P4-P6. We found that derivatives of the ribozyme containing the modules performed the reversal of the first step of the self-splicing reaction efficiently by using their affinity to the substrate RNA, although their kcat values and substrate specificity were uninfluenced and limited, respectively. The results show that it is possible to add a variety of new domains at the peripheral region that play a role comparable with that of the conserved P4-P6 domain.

Base Sequence↗
PubMed2003

Biochemical characterization of the kink-turn RNA motif.

RNA, which acts as a medium for transmitting genetic information, plays a variety of roles in a cell. As with proteins, elucidation of the three- dimensional (3D) structures of RNAs is important for understanding their various roles. Determination of the atomic structures of crystallized ribosome has enabled the identification of previously unknown RNA structural motifs. The kink-turn (K-turn or GA) motif, which causes a sharp bend in an RNA double helix, was identified as one of these structural motifs. To biochemically characterize the K-turn, the motif was inserted into a hinge region of P4-P6 RNA, which is the most extensively studied self-folding RNA, and its properties were investigated. The stability and metal ion requirement of the constructs containing three different K-turn motifs were analyzed using native PAGE and dimethyl sulfate (DMS) modification. The formation of the sharp bending structure depends on the presence of divalent cation like Mg2+ or Ca2+, although its required concentration is different for each motif.

Base Pairing↗
PubMed2003

Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP.

A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA-protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA-RNA interaction in the starting ribozyme is replaced with an RNA-protein interaction via two intermediary stages. At each stage, the original RNA-RNA interaction and a newly introduced RNA-protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA-RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.

Animals↗
PubMed2003

Selections for constituting new RNA-protein interactions in catalytic RNP.

In vitro and in vivo selection techniques are developed to constitute new RNA-peptide interactions. The selection strategy is designed by employing a catalytic RNP consisting of a derivative of the Tetrahymena ribozyme and an artificial RNA-binding protein. An arginine-rich RNA-binding motif and its target RNA motif in the RNP are substituted with randomized sequences and used for the selection experiments. Previously unknown binding motifs are obtained and the newly established interactions have been indispensable for assembling a catalytically active RNP. The method employed in this study is useful for making customized self-splicing intron RNAs whose activity is regulated by protein cofactors.

Binding Sites↗
PubMed2003

Designed structural-rearrangement of an active group I ribozyme.

The catalytic core of the Tetrahyemena group I ribozyme consists of two functionally different domains, P4-P6 and P3-P7, that are conjugated via multiple tertiary interactions. The sequence encoding the P3-P7 domain is divided into two fragments in its primary sequence although the two domains are physically separable in the three dimensional (3D-) structure of the ribozyme: The sequence encoding the P4-P6 domain is inserted into that of the P3-P7 domain. An artificial rearrangement was designed and attempted for the primary sequence of the P3-P7 domain on the basis of a 3D-structural model and the biochemical data on the ribozyme. The domain in the primary structure was relocated to form a contiguous region while retaining the 3D-structure of the ribozyme required for self-splicing. The topologically rearranged ribozyme exhibited self-splicing activity.

Animals↗
PubMed2003

The P5 activator of a group IC ribozyme can replace the P7.1/7.2 activator of a group IA ribozyme.

The P5 or P7 extensions in the group I intron ribozyme serve as "modular activator units" by stabilizing the conserved core of the ribozyme. The P5 extension of a group IC1 intron was introduced to a barely active group IA2 intron lacking its original P7 extension. The inserted P5 extension significantly activated the chimeric construct. Because the CYT-18 protein factor is also known to activate mutant group IA2 and IC1 introns lacking their P7 and P5 extensions, respectively, the RNA and protein activator units function in an analogous manner.

Alternative Splicing↗
PubMed2002

Modular engineering of a Group I intron ribozyme.

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4-P6 and P3-P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3-P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3-P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3-P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.

Base Sequence↗
PubMed2002

Relationship between the self-splicing activity and the solidity of the master domain of the Tetrahymena group I ribozyme.

The highly conserved P3-P7 domain of the Group I intron ribozymes is known to contain essential elements, such as the binding site for the cofactor guanosine, required for conducting the splicing reaction. We investigated the domain of the Tetrahymena intron ribozyme and its variants in order to clarify the relationship between its stability and function. We found that the destabilization of the P3-P7 domain facilitates the active structure formation at high magnesium ion concentrations where the formation is retarded for the wild type. The destabilized domain also increases K(GTP)(m) although this can be compensated by increasing the concentration of Mg(2+), indicating that the stable domain is required for establishing a tight guanosine binding site. The results suggest that the stability of the domain affects the rate-limiting step in the RNA folding pathway and also regulates the efficiency of the splicing reaction.

Animals↗
PubMed2002

Design, construction, and analysis of a novel class of self-folding RNA.

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.

Animals↗
PubMed2002

Mispaired P3 region in the hierarchical folding pathway of the Tetrahymena ribozyme.

BACKGROUND: The Tetrahymena group I ribozyme folds into a complex three-dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4-P6 and P3-P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate. RESULTS: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt-P3) appears a likely candidate. To examine the effects of the alt-P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3-P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt-P3 structure were altered systematically. CONCLUSION: The results indicate that the alt-P3 structure is not the major rate-limiting factor in the folding process.

Animals↗