Yohei Yokobayashi
University of California, Davis
Network
Latest external collaboration on country level. Dive into details by clicking on the dots.
Publication
Featured researches published by Yohei Yokobayashi.
Proceedings of the National Academy of Sciences of the United States of America | 2002
Yohei Yokobayashi; Ron Weiss; Frances H. Arnold
The construction of artificial networks of transcriptional control elements in living cells represents a new frontier for biological engineering. However, biological circuit engineers will have to confront their inability to predict the precise behavior of even the most simple synthetic networks, a serious shortcoming and challenge for the design and construction of more sophisticated genetic circuitry in the future. We propose a combined rational and evolutionary design strategy for constructing genetic regulatory circuits, an approach that allows the engineer to fine-tune the biochemical parameters of the networks experimentally in vivo. By applying directed evolution to genes comprising a simple genetic circuit, we demonstrate that a nonfunctional circuit containing improperly matched components can evolve rapidly into a functional one. In the process, we generated a library of genetic devices with a range of behaviors that can be used to construct more complex circuits.
Nucleic Acids Research | 2009
Norihito Muranaka; Vandana Sharma; Yoko Nomura; Yohei Yokobayashi
Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.
Journal of the American Chemical Society | 2009
Deepak Kumar; Chung Il An; Yohei Yokobayashi
Conditional RNA interference (RNAi) enables spatial and/or temporal control over gene silencing. The currently available methods require coexpression of engineered proteins and/or modified promoters which may limit their applications. We designed a novel RNA architecture that combines a drug-inducible allosteric ribozyme with a microRNA precursor analogue that allows chemical induction of RNAi in mammalian cells. The compact and highly modular RNA design should facilitate the construction of conditional RNAi systems that can sense and respond to a variety of molecules recognized by RNA aptamers to regulate virtually any desired genes sensitive to RNAi.
Chemical Communications | 2007
Wataru Yoshida; Yohei Yokobayashi
We designed a pair of DNA-based logic gates that sense single-stranded DNAs and aptamer ligands to produce fluorescence outputs according to Boolean logic functions AND and OR.
ACS Synthetic Biology | 2013
Yoko Nomura; Linlin Zhou; Anh Miu; Yohei Yokobayashi
We engineered small molecule responsive allosteric ribozymes based on the genomic hepatitis delta virus (HDV) ribozyme by replacing the P4-L4 stem-loop with an RNA aptamer through a connector stem. When embedded in the 3′ untranslated region of a reporter gene mRNA, these RNA devices enabled regulation of cis-gene expression by theophylline and guanine by up to 29.5-fold in mammalian cell culture. Furthermore, a NOR logic gate device was constructed by placing two engineered ribozymes in tandem, demonstrating the modularity of the RNA devices. The significant improvement in the regulatory dynamic range (ON/OFF ratio) of the RNA devices based on the HDV ribozyme should provide new opportunities for practical applications.
Biochemical and Biophysical Research Communications | 2008
Nazgul Tuleuova; Chung Il An; Erlan Ramanculov; Alexander Revzin; Yohei Yokobayashi
RNA interference (RNAi) has emerged as a powerful technology to silence arbitrary genes by designing small RNA constructs based on the targeted messenger RNA sequences. We recently developed a small molecule-controlled RNAi gene switch that combined the molecular recognition by in vitro selected RNA aptamers with versatile gene silencing by small interfering RNAs, and demonstrated for the first time, posttranscriptional modulation of RNAi through direct RNA-small molecule interaction. In this report, we describe the first application of this technology to regulate an endogenous gene in mammalian cells. As a proof-of-concept demonstration we chose to modulate expression of albumin-serum protein produced by the liver. We designed and constructed a theophylline aptamer-fused short hairpin RNA (shRNA) expression vector targeting albumin mRNA in hepatic (HepG2) cells. Transfection of HepG2 cells with the aptamer-shRNA expression vector allowed to control albumin gene expression by adding theophylline into the culture media in dose dependent fashion.
Chemical Communications | 2012
Yoko Nomura; Deepak Kumar; Yohei Yokobayashi
Allosteric hammerhead ribozymes (aptazymes) that are activated by guanine were used to control mammalian gene expression in cis and in trans. Coexpression of the two mechanistically distinct riboswitches resulted in an improved dynamic range of gene expression.
ChemBioChem | 2009
Norihito Muranaka; Koichi Abe; Yohei Yokobayashi
After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism‐guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo‐Shine‐Dalgarno (SD) sequence located near the 3′‐end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamers P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions.
Current Opinion in Biotechnology | 2003
Cynthia H. Collins; Yohei Yokobayashi; Daisuke Umeno; Frances H. Arnold
Recent protein engineering efforts have generated artificial transcription factors that bind new target DNA sequences and enzymes that modify DNA at new target sites. Zinc-finger-based transcription factors are favored targets for design; important technological advances in their construction and numerous biotechnological applications have been reported. Other notable advances include the generation of endonucleases and recombinases with altered specificities, made by innovative combinatorial and evolutionary protein engineering strategies. An unexpectedly high tolerance to mutation in the active sites of DNA polymerases is being exploited to engineer polymerases to incorporate artificial nucleotides or to display other, nonnatural activities.
BioSystems | 2007
Yoko Nomura; Yohei Yokobayashi
Forward engineering of synthetic genetic circuits in living cells is expected to deliver various applications in biotechnology and medicine and to provide valuable insights into the design principles of natural gene networks. However, lack of biochemical data and complexity of biological environment complicate rational design of such circuits based on quantitative simulation. Previously, we have shown that directed evolution can complement our weakness in designing genetic circuits by screening or selecting functional circuits from a large pool of nonfunctional ones. Here we describe a dual selection strategy that allows selection of both ON and OFF states of genetic circuits using tetA as a single selection marker. We also describe a successful demonstration of a genetic switch selection from a 2000-fold excess background of nonfunctional switches in three rounds of iterative selection. The dual selection system is more robust than the previously reported selection system employing three genes, with no observed false positive mutants during the simulated selections.