Photonic Boolean logic gates based on DNA aptamers.
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.
Adenosine↗
Biomedical subjects
Yohei Yokobayashi
Publications and source records attributed to Yohei Yokobayashi.
Dual selection of a genetic switch by a single selection marker.
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.
Computer Simulation↗
Artificial control of gene expression in mammalian cells by modulating RNA interference through aptamer-small molecule interaction.
Recent studies have uncovered extensive presence and functions of small noncoding RNAs in gene regulation in eukaryotes. In particular, RNA interference (RNAi) has been the subject of significant investigations for its unique role in post-transcriptional gene regulation and utility as a tool for artificial gene knockdown. Here, we describe a novel strategy for post-transcriptional gene regulation in mammalian cells in which RNAi is specifically modulated through RNA aptamer-small molecule interaction. Incorporation of an RNA aptamer for theophylline in the loop region of a short hairpin RNA (shRNA) designed to silence fluorescent reporter genes led to dose-dependent inhibition of RNAi by theophylline. shRNA cleavage experiments using recombinant Dicer demonstrated that theophylline inhibited cleavage of an aptamer-fused shRNA by Dicer in vitro. Inhibition of siRNA production by theophylline was also observed in vivo. The results presented here provide the first evidence of specific RNA-small molecule interaction affecting RNAi, and a novel strategy to regulate mammalian gene expression by small molecules without engineered proteins.
Aptamers, Nucleotide↗
Exploration of structural features of monomeric helical peptides designed with a genetic algorithm.
A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.
Algorithms↗
Engineering proteins that bind, move, make and break DNA.
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DNA↗
Engineering proteins that bind, move, make and break DNA.
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.
DNA↗
Directed evolution of a genetic circuit.
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.
DNA↗