Riboregulator elements as tools to engineer gene expression in cyanobacteria.

Cyanobacteria are an ideal host for biofuel production. Although efforts have been made to genetically engineer cyanobacteria for efficient production of biofuels and other important chemicals, the tools that can be applied to cyanobacteria are still limited. A new gene regulation tool, riboregulator, has been examined for application in cyanobacteria. A riboregulator is a nature-inspired RNA tool, which is composed of two artificially designed RNA fragments. Owing to its high specificity and efficacy, it is suitable for metabolic engineering in cyanobacteria, and several studies have been done to optimize and improve the function of the riboregulator. In this review, we focus on the recent improvements made to riboregulators and compare them with other RNA-mediated gene regulation tools developed in cyanobacteria to investigate future applications of riboregulators.

Applying a riboregulator as a new chromosomal gene regulation tool for higher glycogen production in Synechocystis sp. PCC 6803.

Cyanobacteria are one of the most attractive hosts for biofuel production; however, genetic approaches to regulate specific chromosomal genes in cyanobacteria remain limited. With the aim of developing a novel method to regulate chromosomal gene expression in cyanobacteria, we focused on riboregulatory technology. Riboregulators are composed of two RNA fragments whose interaction leads to target gene regulation with high specificity. In this study, we inserted a riboregulator sequence upstream of the chromosomal gene encoding AbrB-like transcriptional regulator, cyAbrB2, to investigate the utility of this tool. The inserted riboregulator was able to regulate cyabrB2 gene expression, with a high ON-OFF ratio up to approximately 50-fold. The transcription levels of several genes for which cyAbrB2 acts as a transcriptional upregulator were also decreased. Further, the cyAbrB2 expression-repressed mutant showed high glycogen accumulation, equivalent to that in the cyabrB2 deletion mutant (ΔcyabrB2). Phenotypic similarities between the cyabrB2 expression-repressed mutant and the ΔcyabrB2 mutant suggest that the riboregulator can potentially be used as a new chromosomal gene regulation tool in cyanobacteria.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (KD = 44 pM) than at pH 7.4 (KD = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.

Data on G-quadruplex topology, and binding ability of G-quadruplex forming sequences found in the promoter region of biomarker proteins and those relations to the presence of nuclear localization signal in the proteins.

Aptamer is a nucleic acid ligand which specifically binds to its target molecule. Previously, we have designed an identification method of aptamer called "G-quadruplex (G4) promoter-derived aptamer selection (G4PAS)" [1]. In G4PAS procedure, putative G4 forming sequences (PQS) were explored in a promoter region of a target protein in human gene through computational analysis, and evaluated binding ability towards the gene product encoded in the downstream of the promoter. We investigated the topology of the obtained PQSs by circular dichroism measurement, as well as their binding ability against its target protein by surface plasmon resonance measurement and gel-shift assay. Additionally, the presence of nuclear localization signal in the target protein was predicted in silico. This data set summarized all the PQS sequences, their biochemical characteristics, and the presence of nuclear localization signal to address the possibility of binding of these PQS region to the target proteins in vivo. Those data should contribute to increase the success rate of G4PAS. Moreover, considering the G4 motifs in genomic DNA are suggested to be involved in vivo gene regulation [2], [3], this data set is also potentially beneficial for the cell biology field.