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Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D ‐Threoninol
Author(s) -
Liang Xingguo,
Fujioka Kenta,
Asanuma Hiroyuki
Publication year - 2011
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201100215
Subject(s) - dna ligase , ligation , azobenzene , dna , moiety , linker , sticky and blunt ends , tethering , sequencing by ligation , chemistry , molecule , base pair , biophysics , stereochemistry , combinatorial chemistry , biochemistry , biology , microbiology and biotechnology , base sequence , organic chemistry , computer science , genomic library , operating system
Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D ‐threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90 % in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320–380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis,.