Robust Enzymatic Production of DNA G-Quadruplex, Aptamer, DNAzyme, and Other Oligonucleotides: Applications for NMR

Single-stranded DNA (ssDNA) oligonucleotides are widely used in biological research, therapeutics, biotechnology, and nanomachines. Large-scale enzymatic production of ssDNA oligonucleotides forming noncanonical structures has been difficult. Here, we present a simple and robust method named “palindrome-nicking-dependent amplification” (PaNDA) for enzymatic production of a large amount of ssDNA oligonucleotides. It utilizes a strand-displacing DNA polymerase and a nicking enzyme together with input DNA and deoxynucleotide triphosphates at 55 °C. Scaling up of PaNDA is straightforward due to its isothermal nature. The ssDNA products can easily be isolated through anion-exchange chromatography under nondenaturing conditions. We demonstrate applications of PaNDA to ¹³C/¹⁵N-labeling of various DNA strands, including a 22-nt telomere repeat G-quadruplex, a 26-nt therapeutic aptamer, and a 33-nt DNAzyme. The ¹³C/¹⁵N-labeling by PaNDA greatly facilitates the characterization of noncanonical DNA by nuclear magnetic resonance (NMR) spectroscopy. For example, the behavior of therapeutic DNA aptamers in human serum can be investigated.