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Design of reactive-end DNA oligomers via incorporation of oxanine into oligonucleotides using terminal deoxynucleotidyl transferase

Authors
Jang, Eui KyoungKi, Mi-RanPack, Seung Pil
Issue Date
11월-2017
Publisher
ELSEVIER SCI LTD
Keywords
Oxanine; Bioconjugation; Terminal deoxynucleotidyl transferase; Reactive-end ODNs
Citation
PROCESS BIOCHEMISTRY, v.62, pp.99 - 105
Indexed
SCIE
SCOPUS
Journal Title
PROCESS BIOCHEMISTRY
Volume
62
Start Page
99
End Page
105
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/81818
DOI
10.1016/j.procbio.2017.07.011
ISSN
1359-5113
Abstract
Oxanine (Oxa, O), a modified nucleobase, has a novel O-acylisourea structure. Oxa-incorporated oligo-deoxynucleotides (ODNs) are reactive DNA oligomers that permit conjugation with various nucleophilic molecules in an activation-free manner. In this study, we developed a new procedure for enzymatic preparation of reactive end DNA oligomers, using terminal deoxynucleotidyl transferase (TdT), in which a reactive Oxa base is incorporated into the 3'-end of ODNs. One limitation of TdT, an enzyme widely used for end labeling of DNA oligomers, is that it is difficult to control the number of incorporated labels, because it shows template-independent extension with random nucleotides. Notably, TdT showed a rate and efficiency of incorporation of the modified nucleobase, Oxa, different from that of natural bases. We investigated the conditions of TdT-mediated DNA incorporation of Oxa and achieved incorporation of Oxa at the 3'-end of ODNs by optimizing reaction parameters such as temperature and enzyme, cofactor, and substrate concentrations. We also confirmed the reactive functionality of Oxa after incorporation into ODNs by amide bonding conjugation with a polyamine (spermine) under physiological conditions, without need for an additional activation step.
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