Abstract
We report enzymatic phosphorylation and additive-free ligation of DNAs containing unnatural C-nucleotide residues through the action of T4 polynucleotide kinase and T4 DNA ligase. The artificial units are each made up of an alkynyl deoxyribose component and one of the unnatural nucleobases D*, T*, G*, and C*, corresponding-from a viewpoint of hydrogen-bonding patterns-to natural A, T, G, and C, respectively. Phosphorylation progressed quantitatively at the 5'-end in the cases of all of the artificial units in the chimeric DNAs. Ligation also smoothly progressed at the 5'-end in the cases of the D* and G* nucleotide residues, but only negligibly in those of their T* and C* counterparts. Chemical redesign of the last two units successfully improved the ligation efficiency, so that enzymatic ligation worked well for all of the artificial units in every 3'-natural⋅5'-artificial, 3'-artificial⋅5'-natural, and 3'-artificial⋅5'-artificial terminal combination at the nicks.
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