Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.
© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.