Shoji J. Ohuchi

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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)
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)
Artificial riboswitches that respond to the concentrations of intracellular proteins are a promising tool with a variety of applications. They can be designed and engineered using existing RNA aptamers that target proteins. Aptamers are generated via an iterative selection-amplification process, known as systematic evolution of ligands by exponential(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)
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)
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)
An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification(More)
Group I intron ribozymes have a modular architecture and structural elements essential for catalysis. The elements are located in the conserved modular domain P3-P7 that is stabilized by another conserved module, P4-P6. It has been reported that artificial modules can complement the function of the native P4-P6. To exploit the modular architecture of group(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)
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)