Novel enzymatic single-nucleotide modification of DNA oligomer: prevention of incessant incorporation of nucleotidyl transferase by ribonucleotide-borate complex

Abstract

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.

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids

“…13,14 The methods of chemical synthesis of DNA have signicantly facilitated the introduction of desired functionalities onto a specic position, 15 and post-synthetic modication methods are also powerful chemical tools, [16][17][18][19] especially for labeling of DNA targets with large labeling groups incompatible during the chemical synthesis process. [20][21][22] Bioinspired enzymatic postsynthetic labeling technologies have emerged as useful approaches through sequence-or structure-dependent substrate recognition, 23 and chemical modication is an alternative means for site-specic DNA labeling by incorporation of a chemical handle that is inert toward endogenous chemical functional groups but reactive toward a labeling reagent. 24,25 However, despite the remarkable advance of the bioconjugation technologies during the past decades, site-specic chemical modication remains a serious challenge due to its demanding criteria which include adequate reactivity of the labeling reagents toward the target site and high selectivity in biomolecule-compatible solvents such as aqueous buffer.…”

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

“…This activity is of major importance in the diversification of immunoglobulins and T cell receptors in the process of V(D)J recombination of the adaptive immune system via random addition of nucleotides to nicked DNA strands. 4 , 5 TdT’s unique ability to mediate template-independent polymerization has made it a valuable tool in a variety of molecular biology applications including finding strand breaks, 6 modifying DNA oligomers with various NTPs, 7 and identifying DNA damage and epigenetic modifications. 8 Furthermore, the enzyme has proven useful for the generation of polynucleotides of high molecular weight 9 and amphiphilic structures upon extension with BODIPY-dUTP, 10 for detection of DNA and RNA on surfaces, 11 , 12 and immobilization of DNA on solid supports.…”

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

“…The average step-yield is 97.7%, which is comparable with the performance of early phosphorylated amidine DNA synthesis [35,53]. A major challenge in TdT synthesis is the control of the addition of single bases, since TdT enzymes tend to catalyze the addition of multiple bases per cycle [65]. Although the synthesis of ssDNA by TdT is still an emerging strategy, several challenges remain.…”

“…Terminal deoxynucleotide transferase (TdT) is a polymerase that indiscriminately adds deoxynucleotide triphosphates (dNTPs) to the 3 end of an ssDNA, which makes it a natural candidate for enzymatic ssDNA synthesis (Figure 2a) [62,63]. TdT is characterized by low substrate specificity for nucleotides and template-independent polymerization [64], which makes TdT-based ssDNA synthesis methods compatible with various modified nucleotides and convenient subsequent purification [65]. Recent studies have shown that the coupling time of C, G, and T is 1.5 min while that of A is 3 min [53].…”

“…Consequently, there is still no implementation of a practical enzymatic oligonucleotide synthesizer based on TdT, but recent application requirements indicate that this is a promising method. Potentially, TdT can be used to synthesize relatively long chains cheaply and quickly, and has been successfully applied in signal amplification [66], single-nucleotide modified in DNA oligos [65], polymerization of building blocks [67], and chain synthesis for DNA information storage [68].…”