In‑Situ ssDNA Isolation from dsDNA Sources as a Streamlined Pathway to DNA Origami Assembly and Testing
Year: 2026
Type: preprint
Abstract: Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: National Science Foundation, Ohio State University, Case Western Reserve University, National Institutes of Health, Materials Research Science and Engineering Center, Harvard University, National Institute of General Medical Sciences, National Institute of Biomedical Imaging and Bioengineering, Division of Materials Research, Division of Chemical, Bioengineering, Environmental, and Transport Systems, Division of Emerging Frontiers in Research and Innovation
Awards: G2487199047, NSF-ANR MCB/PHY: DNA Origami-based machines for epigenetic manipulation of gene transcription, CAREER: Structural Implications of Nucleic Acid Nanoparticles on their Transcription, Translation, and Transport, EFRI CEE: DNA origami tools to engineer chromatin structure and function in live cells, G3723821865, Center for Emergent Materials, Cytosolic Access and Instability of DNA nanoparticles, G6012531032, Atomic Force Microscope, G6336286406, DMREF/Collaborative Research: DNA-based Sensing, Communicating, and Phase-Separating Materials, G7540121869, Mechanisms of chromatin regulation of transcription
OpenAlex ID: W7140102899