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...
The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which is an adaptive immune system of bacteria, has become a powerful tool for genome editing in various model organisms. Here, we demonstrate multiple genome modifications mediated by CRISPR/Cas9 in zebrafish (Danio rerio). Multiple genes including golden/gol and tyrosinase/tyr, which are involved in pigment formation, and s1pr2 and spns2, which are involved in cardiac development, were disrupted with insertion and/or deletion (indel) mutations introduced by the co-injection of multiple guide RNAs (gRNAs) and the nuclease Cas9 mRNA. We simultaneously observed two distinct phenotypes, such as, the two hearts phenotype and the hypopigmentation of skin melanophores and the retinal pigment epithelium, in the injected F0 embryos. Additionally, we detected the targeted deletion and inversion genes as a 7.1-kb fragment between the two distinct spns2 targeted sites together with indel mutations. Conversely, chromosomal translocations among five target loci were not detected. Therefore, we confirmed that the CRISPR/Cas9-induced indel mutations and a locus-specific deletion were heritable in F1 embryos. To screen founders, we improved heteroduplex mobility assay (HMA) for simultaneously detecting indel mutations in different target loci. The results suggest that the multi-locus HMA is a powerful tool for identification of multiple genome modifications mediated by the CRISPR/Cas9 system.
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.
RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM).
meso-Hexakis(pentafluorophenyl)-substituted neutral hexaphyrin with a 26π-electronic circuit can be regarded as a real homolog of porphyrin with an 18π-electronic circuit with respect to a quite flat molecular structure and strong aromaticity. We have investigated additional aromaticity enhancement of meso-hexakis(pentafluorophenyl)[26]hexaphyrin(1.1.1.1.1.1) by deprotonation of the inner N-H groups in the macrocyclic molecular cavity to try to induce further structural planarization. Deprotonated mono- and dianions of [26]hexaphyrin display sharp B-like bands, remarkably strong fluorescence, and long-lived singlet and triplet excited-states, which indicate enhanced aromaticity. Structural, spectroscopic, and computational studies have revealed that deprotonation induces structural deformations, which lead to a change in the main conjugated π-electronic circuit and cause enhanced aromaticity.
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