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

Mari Takahara, Kounosuke Hayashi, Masahiro Goto, Noriho Kamiya

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)660-665
Number of pages6
JournalJournal of Bioscience and Bioengineering
Volume116
Issue number6
DOIs
Publication statusPublished - Dec 1 2013

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Nucleotide Aptamers
Tailings
DNA
Enzymes
Transglutaminases
Proteins
DNA Nucleotidylexotransferase
Peptides
Dideoxynucleotides
Nucleotides
Glutamine
RNA
Thrombin
Labeling
Lysine
Limit of Detection
Assays
Substrates

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

Tailing DNA aptamers with a functional protein by two-step enzymatic reaction. / Takahara, Mari; Hayashi, Kounosuke; Goto, Masahiro; Kamiya, Noriho.

In: Journal of Bioscience and Bioengineering, Vol. 116, No. 6, 01.12.2013, p. 660-665.

Research output: Contribution to journalArticle

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