Enzymatic conjugation of multiple proteins on a DNA aptamer in a tail-specific manner

Mari Takahara, Kounosuke Hayashi, Masahiro Goto, Noriho Kamiya

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)


Conjugation of single-strand DNA aptamers and enzymes has been of great significance in bioanalytical 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 was 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.

Original languageEnglish
Pages (from-to)814-823
Number of pages10
JournalBiotechnology Journal
Issue number6
Publication statusPublished - Jun 1 2016

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Molecular Medicine


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