biostudies-literatureSun WOpen Foundation of State Key Laboratory of Marine Food Processing & Safety Controlthe National Key R&D Program of ChinaYouth Innovation Team Program of Universities in Shandong Provincethe National Natural Science Foundation of China1027https://www.ebi.ac.uk/biostudies/studies/S-EPMC11352879biostudies-literatureUnknown14(8)The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' 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 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 witBiomoleculesFacile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick.PMC11352879SKL2023082019YFD09017012023KJ04032102064An RLiu MLuo JLiang XHu KSun WfalseFacile Splint-Free Circularization of ssDNA with T4 DNA Ligase by Redesigning the Linear Substrate to Form an Intramolecular Dynamic Nick.The efficient preparation of single-stranded DNA (ssDNA) rings, as a macromolecular construction approach with topological features, has aroused much interest due to the ssDNA rings' 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 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 wit2024-01-01T00:00:00Z2024 Aug2025-04-26T11:42:12.03Z2025-04-06T13:45:30.333ZS-EPMC113528793919941410.3390/biom14081027