Journal
BIOTECHNOLOGY JOURNAL
Volume 11, Issue 6, Pages 814-823Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201500560
Keywords
DNA-enzyme; Microbial transglutaminase; Multiple enzyme assembly; Site-specific conjugation; Terminal deoxynucleotidyl transferase
Funding
- Grants-in-Aid for Scientific Research [14J04260] Funding Source: KAKEN
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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.
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