Tailing DNA aptamers with a functional protein by two-step enzymatic reaction

详细信息    查看全文

• 作者：Mari Takahara ; Kounosuke Hayashi ; Masahiro Goto ; Noriho Kamiya
• 关键词：Enzyme-linked aptamer assay ; DNA aptamer ; DNA-enzyme conjugate ; Microbial transglutaminase ; Thrombin ; Terminal deoxynucleotidyl transferase
• 刊名：Journal of Bioscience and Bioengineering
• 出版年：December, 2013
• 年：2013
• 卷：116
• 期：6
• 页码：660-665
• 全文大小：866 K

文摘

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鈥?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.