biostudies-literatureWang XWelch FoundationJapan Science and Technology AgencyNational Institute of General Medical SciencesNIGMS NIH HHS1748-1752https://www.ebi.ac.uk/biostudies/studies/S-EPMC10926321biostudies-literatureUnknown146(3)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.Journal of the American Chemical SocietyRobust Enzymatic Production of DNA G-Quadruplex, Aptamer, DNAzyme, and Other Oligonucleotides: Applications for NMR.PMC10926321R35-GM130326JPMJFS2125R35 GM130326H-2104-20220331Paz-Villatoro JMYu BIwahara JSakurabayashi SWang XfalseRobust 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.2024-01-01T00:00:00Z2024 Jan2025-04-03T23:36:16.939Z2025-04-03T23:36:16.939ZS-EPMC109263213819199310.1021/jacs.3c11219