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A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then(More)
Aptamers are molecules identified from large combinatorial nucleic acid libraries by their high affinity to target molecules. Due to a variety of desired properties, aptamers are attractive alternatives to antibodies in molecular biology and medical applications. Aptamers are identified through an iterative selection-amplification process known as(More)
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(More)
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,(More)
We have previously reported that a protein library can be constructed by directly combining PCR amplification of a single DNA molecule and cell-free protein synthesis. To specifically amplify single DNA molecules, however, two-step PCR with nested primers was used. Here we describe a simpler method for single-step amplification of a single molecule. The(More)
Multifunctional molecular complexes are valuable tools with a variety of applications. We have developed an RNA-protein complex (RNP) containing three different proteins attached to the tips of a triangular RNA scaffold. We designed and constructed three RNA strands that specifically bind a ribosomal protein, L7Ae, and that autonomously form a single(More)
RNA aptamers are one of the promising components for constructing artificial genetic circuits. In this study, we developed a transcriptional activator based on an RNA aptamer against one of the most frequently applied repressor proteins, lambda phage cI. In vitro selection (Systematic Evolution of Ligands by EXponential enrichment) and following in vivo(More)
Catalysis of Group I intron ribozymes is carried out by its core region consisting of two helical domains P4-P6 and P3-P7. Recently, our laboratory showed that a mutant Group I ribozyme lacking both the P4-P6 domain and the base-triples can perform the trans-esterification reactions. The result demonstrates that the elements required for splicing are(More)
RNA-protein complexes (RNPs) are useful for constructing functional nano-objects because a variety of functional proteins can be displayed on a designed RNA scaffold. Here, we report circular permutations of an RNA-binding protein L7Ae based on the three-dimensional structure information to alter the orientation of the displayed proteins on the RNA(More)
In this report, we describe the development of a novel in vitro high-throughput system for detecting and screening promoter activity; the method employs emulsified reactions and a ligase ribozyme. In our study, a promoter DNA fragment containing the ribozyme gene was immobilized on a bead by using emulsion PCR, followed by in vitro transcription of the(More)