Web of Science

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQEGFP) and a primary amine clustered DNA aptamer was enzymatically synthesized as a novel aryl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form.

DOI： 10.1021/acs.bioconjchem.7b00594

Web of Science

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

A novel artificial cellulase was developed by conjugating a DNA aptamer to an endoglucanase catalytic domain, thereby substituting the natural carbohydrate-binding module. Circular dichroism spectroscopy and adsorption isotherm showed the binding motif of cellulose-binding DNA aptamer (CelApt) was G-quadruplex and stem-loop structures stabilized in the presence of salts, and CelApt binding preferred the amorphous region of the solid cellulose. By introducing the revealed salt-switchable cellulose binding nature of CelApt into a catalytic domain of a cellulase, we created CelApt-catalytic domain conjugate possessing both controllable adsorption on the solid substrates and equal enzymatic activity to the wild-type cellulase. Thus potential use of a responsive DNA aptamer for biocatalysis at a solid surface was demonstrated.

DOI： 10.1021/acs.biomac.6b01141

Web of Science

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-V C H VERLAG GMBH  

Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bio-analytical and biomedical applications because of the unlimited functions provided by DNA aptamer direction. Therefore, we developed efficient tailing of a DNA aptamer, with end-specific conjugation of multiple enzymes, through enzymatic catalysis. Terminal deoxynucleotidyl transferase (TdT) added multiple Z-Gln-Gly (Z-QG) moieties to the 3'-end of a DNA aptamer via the addition of Z-QG-modified deoxyuridine triphosphate (Z-QG-dUTP) and deoxynucleoside triphosphates (dNTPs). The resultant (Z-QG)(m)-(dN)(l)-aptamer, whose Z-QGs with dN spacers served as stickers for microbial transglutaminase (MTG), were crosslinked between the Z-QGs on the DNA and a substrate peptide sequence containing lysine (K), fused to a recombinant enzyme (i.e. bacterial alkaline phosphatase; BAP) by MTG. The incorporation efficiency of Z-QG moieties on the aptamer tail and the subsequent conjugation efficiency with multiple enzyme molecules were dramatically altered by the presence of dNTPs, revealing that a combination of Z-QG-dUTP/dTTP comprised the best labeling efficiency and corresponding properties during analytical performance. Thus, a novel optimized platform for designing (BAP)(n)-(dT)(l)-DNA aptamers is demonstrated for the first time in this article, offering unique opportunities for tailoring new types of covalent protein-nucleic acid conjugates in a controllable way.

DOI： 10.1002/biot.201500560

Web of Science

J-GLOBAL

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

An efficient, quantitative synthetic strategy for aptamer-enzyme conjugates was developed by using a two-step enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT) was used to first incorporate a Z-Gln-Gly (QG) modified nucleotide which can act as a glutamine donor for a subsequent enzymatic reaction, to the 3'-OH of a DNA aptamer. Microbial transglutaminase (MTG) then catalyzed the cross-linking between the Z-QG modified aptamers and an enzyme tagged with an MTG-reactive lysine containing peptide. The use of a Z-QG modified dideoxynucleotide (Z-QG-ddUTP) or a deoxyuridine triphosphate (Z-QG-dUTP) in the TdT reaction enables the controlled introduction of a single or multiple MTG reactive residues. This leads to the preparation of enzyme-aptamer and (enzyme)n-aptamer conjugates with different detection limits of thrombin, a model analyte, in a sandwich enzyme-linked aptamer assay (ELAA). Since the combination of two enzymatic reactions yields high site-specificity and requires only short peptide substrates, the methodology should be useful for the labeling of DNA/RNA aptamers with proteins. © 2013 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2013.05.025

Scopus

PubMed

J-GLOBAL

researchmap

記述言語：日本語   出版者・発行元：日本生物工学会  

CiNii Article

CiNii Books

researchmap

その他リンク： http://search.jamas.or.jp/link/ui/2014247461

開催年月日： 2020年8月17日 - 2020年8月20日

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：ポスター発表  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：ポスター発表  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（指名）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：ポスター発表  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：日本語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：ポスター発表  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap

記述言語：英語   会議種別：口頭発表（一般）  

researchmap