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As the interest in DNA nanotechnology increases, so does the need for larger and more complex DNA structures. In this work, we describe two methods of using large, double-stranded (ds) DNA to self-assemble sequence-specific, nonrepetitive microscale structures. A model system restructures T7 DNA (40 kb) through sequence-specific biotinylation followed by intramolecular binding to a 40 nm diameter neutravidin bead to create T7 "rosettes". This model system informed the creation of "nodal DNA" where "nodes" with single-stranded DNA flaps are attached to a large dsDNA insert so that a complementary oligonucleotide "strap" bridges the two nodes for restructuring to form a DNA "bolo". To do this in high yield, several methodologies were developed, including a protection/deprotection scheme using RNA/RNase H and dialysis chambers, which remove excess straps while retaining large DNA molecules. To assess these restructuring processes, the DNA was adsorbed onto supported lipid bilayers, allowing for a visual assay of their structure using single-molecule fluorescence microscopy. Good agreement between the expected and observed fluorescence intensity measurements of the individual features of restructured DNA for both the DNA rosettes and bolos gives us a high degree of confidence that both processes give sequence-specific restructuring of large, dsDNA molecules to create microscale objects.

Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules