Momoko Kitaoka

Transglutaminase‐Mediated Synthesis of a DNA–(Enzyme)n Probe for Highly Sensitive DNA Detection

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.

Aligning an endoglucanase Cel5A from Thermobifida fusca on a DNA scaffold: potent design of an artificial cellulosome

A novel multi-cellulase conjugate assembled on a double-stranded DNA scaffold, a DNA-(endoglucanase)n conjugate, exhibited unique hydrolytic activity toward crystalline cellulose (Avicel) depending on the cellulase/DNA ratio on the DNA-based artificial cellulosome.

Mutation Detection in DNA Oligonucleotides Based on a Guanine Quenching Method Coupled with Enzymatic Digestion of Single-Stranded DNA

Fluorescence quenching by guanine allows DNA hybridization to be monitored and any point mutations in oligonucleotides to be detected. However, fluorescence quenching is often affected by untargeted guanine located in a protruding end (single-strand DNA) of the probe-target DNA duplex resulting in an unsatisfactory sensitivity. In the present study, we used enzymatic digestion of the protruding end of a probe-target DNA duplex to avoid interference by untargeted guanine on fluorescence quenching for detection of a nucleobase mutation. Enzymatic digestion of the protruding end of the DNA duplex fully prevented interference by untargeted guanine, and produced a marked difference in the quenching ratios (36% for wild-type, and 0% for mutant).

Programmable protein–protein conjugation via DNA-based self-assembly

Protein molecules were precisely arrayed on a designable DNA scaffold close to each other using a DNA aptamer. By adding a chemical cross-linker, the neighboring protein molecules were effectively and covalently cross-linked to each other without losing their activities.

Sucrose laurate-enhanced transcutaneous immunization with a solid-in-oil nanodispersion

A novel transcutaneous immunization system was developed using a solid-in-oil (S/O) nanodispersion, consisting of a nano-sized particle of a protein–surfactant complex dispersed in an oil vehicle. Permeability of the model antigen protein, ovalbumin, was enhanced by coating with sucrose laurate, which is a hydrophobic surfactant. The nanodispersion prepared with sucrose laurate induced a 5-fold increase in mouse antigen-specific antibody production compared with that observed using sucrose erucate, indicating that stability of the S/O nanodispersion in the epidermis plays a key role in enabling effective immunization. In spite of the coating with the surfactants, only fluorescence-labeled ovalbumin permeated the deep epidermis beneath the stratum corneum and was observed by laser scanning confocal microscopy. These findings will lead the way to an improved transcutaneous immunization system based on S/O nanodispersion.

Needle-free immunization using a solid-in-oil nanodispersion enhanced by a skin-permeable oligoarginine peptide.

The objective of transcutaneous immunization is efficient vaccination using the skins immune system. Although a less invasive administration procedure is involved, the effective delivery of antigen using this modality remains a problem. Here, we demonstrate the use of a solid-in-oil (S/O) nanodispersion system for the transcutaneous immunization of male ddY mice with ovalbumin (OVA) antigen. The S/O nanoparticles consisted of OVA and hydrophobic surfactant molecules and were dispersed in an oil; enhanced induction of antigen-specific antibody was observed after the addition of polyarginine (R6) into the same S/O nanoparticle containing OVA. The improved S/O nanodispersion system induced a comparable level of OVA-specific antibody to that induced by subcutaneous injection of OVA at the same dose, advocating the potential application of the S/O system as a needle-free and easy-to-use immunization system.

Transglutaminase-mediated in situ hybridization (TransISH) system: a new methodology for simplified mRNA detection.

Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen-antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection.

Microplate assay for aptamer-based thrombin detection using a DNA–enzyme conjugate based on histidine-tag chemistry

We report a method to prepare a DNA-enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose K(d) was approximately 10⁻⁶ (M⁻¹) toward a His-tag present on a recombinant protein via the complexation of Ni²⁺. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni²⁺ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni²⁺ complex. The DNA-enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized.

Solid-in-oil nanodispersions for transdermal drug delivery systems.

Transdermal administration of drugs has advantages over conventional oral administration or administration using injection equipment. The route of administration reduces the opportunity for drug evacuation before systemic circulation, and enables long‐lasting drug administration at a modest body concentration. In addition, the skin is an attractive route for vaccination, because there are many immune cells in the skin. Recently, solid‐in‐oil nanodisperison (S/O) technique has demonstrated to deliver cosmetic and pharmaceutical bioactives efficiently through the skin. S/O nanodispersions are nanosized drug carriers designed to overcome the skin barrier. This review discusses the rationale for preparation of efficient and stable S/O nanodispersions, as well as application examples in cosmetic and pharmaceutical materials including vaccines. Drug administration using a patch is user‐friendly, and may improve patient compliance. The technique is a potent transcutaneous immunization method without needles.

Transdermal Immunization using Solid-in-oil Nanodispersion with CpG Oligodeoxynucleotide Adjuvants

PurposeSimple and noninvasive vaccine administration alternatives to injections are desired. A solid-in-oil (S/O) nanodispersion system was able to overcome skin barriers and induce an immune response; however, antibody levels remained low. We applied an immune potentiator, CpG oligodeoxynucleotide (ODN), to enhance the immune response by controlling the T helper 1 (Th1)/T helper 2 (Th2) balance.MethodsS/O nanodispersions containing ovalbumin (OVA) and CpG ODN (CpG-A or CpG-B) were characterized by size distribution analysis and a protein release test. The skin permeation of fluorescence-labeled OVA was observed by fluorescence microscopy. Antigen-specific IgG, IgG1, and IgG2a responses were measured by enzyme-linked immunosorbent assay.ResultsCo-encapsulation of CpG ODNs in S/O nanodispersions enhanced induction of OVA-specific IgG. S/O nanodispersion containing OVA and CpG-A had a smaller mean particle size and permeated the skin more efficiently. In contrast, CpG-B showed the highest protein release and induction of OVA-specific IgG. IgG subclass analysis revealed that OVA induced a Th2-dominant immune response, while the S/O nanodispersion containing CpG-A skewed the immune response toward a Th1-bias.ConclusionsIn combination with CpG ODN, the S/O nanodispersion system efficiently induced an antigen-specific antibody response. The Th1/Th2 immune balance could be controlled by the selection of CpG ODN type.