Version 1
: Received: 1 August 2024 / Approved: 2 August 2024 / Online: 5 August 2024 (05:21:41 CEST)
How to cite:
Sun, W.; Hu, K.; Liu, M.; Luo, J.; An, R.; Liang, X. Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick. Preprints2024, 2024080191. https://doi.org/10.20944/preprints202408.0191.v1
Sun, W.; Hu, K.; Liu, M.; Luo, J.; An, R.; Liang, X. Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick. Preprints 2024, 2024080191. https://doi.org/10.20944/preprints202408.0191.v1
Sun, W.; Hu, K.; Liu, M.; Luo, J.; An, R.; Liang, X. Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick. Preprints2024, 2024080191. https://doi.org/10.20944/preprints202408.0191.v1
APA Style
Sun, W., Hu, K., Liu, M., Luo, J., An, R., & Liang, X. (2024). Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick. Preprints. https://doi.org/10.20944/preprints202408.0191.v1
Chicago/Turabian Style
Sun, W., Ran An and Xingguo Liang. 2024 "Facile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick" Preprints. https://doi.org/10.20944/preprints202408.0191.v1
Abstract
The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to their numerous applications in biotechnology and DNA nanotechnology. However, an extra splint is essential for enzymatic circularization, and by-products of multimers are usually present at high concentrations. Here, we proposed a simple and robust strategy of using permuted precursor (linear ssDNA) for circularization by forming an intramolecular dynamic nick using a part of the linear ssDNA substrate itself as the template. After the simulation of the secondary structure for desired circular ssDNA, the linear ssDNA substrate is designed to have its ends on the duplex part (≥5 bp). By using this permuted substrate with 5'-phosphate, the splint-free circularization is simply carried out by T4 DNA ligase. Very interestingly, formation of only several base pairs (2–4) flanking the nick is enough for ligation, although they form only instantaneously under ligation conditions. More significantly, the 5 bp intramolecular duplex part commonly exists in genomes or functional DNA, demonstrating the high generality of our approach. Our findings are helpful for understanding the mechanism of enzymatic DNA ligation from the viewpoint of substrate binding.
Keywords
ssDNA circularization; permuted sequence; T4 DNA ligase; splint-free ligation
Subject
Chemistry and Materials Science, Biomaterials
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
