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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 “palind...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2024-01, Vol.146 (3), p.1748-1752
Main Authors: Wang, Xi, Yu, Binhan, Sakurabayashi, Shuhei, Paz-Villatoro, Jonathan M., Iwahara, Junji
Format: Article
Language:English
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Summary: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.
ISSN:0002-7863
1520-5126
1520-5126
DOI:10.1021/jacs.3c11219