Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent sensitivity.

Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity.

Site-specific functionalization of RNA molecules by an unnatural base pair transcription system via click chemistry.

Toward new biotechnology by genetic alphabet expansion, we developed an efficient site-specific labeling method for large RNA molecules. The combination of unnatural base pair transcription and post-transcriptional modification by click chemistry enables simple RNA labeling with a wide variety of functional groups at desired positions, in a one-pot reaction.

Unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma.

Toward the expansion of the genetic alphabet of DNA, several artificial third base pairs (unnatural base pairs) have been created. Synthetic DNAs containing the unnatural base pairs can be amplified faithfully by PCR, along with the natural A-T and G-C pairs, and transcribed into RNA. The unnatural base pair systems now have high potential to open the door to next generation biotechnology.

PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-unnatural-base-pair system.

For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase.

Highly specific unnatural base pair systems as a third base pair for PCR amplification.

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity.

Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system.

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification.

Unnatural base pair systems for sensing and diagnostic applications.

Expansion of the genetic alphabet by an unnatural base pair system provides a platform for the site-specific, enzymatic incorporation of extra, functional components into nucleic acids. Recently, several unnatural base pairs that exhibit high fidelity and efficiency in PCR have been developed. Functional groups of interest, such as fluorescent dyes, can be linked to the unnatural bases, and the modified base substrates are site-specifically incorporated into nucleic acids by polymerases.

A unique fluorescent base analogue for the expansion of the genetic alphabet.

Fluorescent nucleobase analogues are useful in a wide variety of biology and biotechnology tools as molecular probes and reporters for nucleic acids. Here we present a novel fluorescent purine analogue, 7-(2,2'-bithien-5-yl)-imidazo[4,5-b]pyridine (denoted as Dss). The nucleoside triphosphates of Dss can be site-specifically incorporated into DNA and RNA by polymerases, opposite its pairing partner, pyrrole-2-carbaldehyde (Pa), in DNA templates.

Automated system for high-throughput protein production using the dialysis cell-free method.

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane.