%0 Online Multimedia %A Krerowicz, Samuel J. W. %A Hernandez-Ortiz, Juan P. %A Schwartz, David C. %D 2018 %T Microscale Objects via Restructuring of Large, Double-Stranded DNA Molecules %U https://acs.figshare.com/articles/media/Microscale_Objects_via_Restructuring_of_Large_Double-Stranded_DNA_Molecules/7361072 %R 10.1021/acsami.8b18157.s005 %2 https://acs.figshare.com/ndownloader/files/13601711 %K nonrepetitive microscale structures %K RNA %K Double-Stranded DNA Molecules %K DNA nanotechnology increases %K fluorescence intensity measurements %K model system %K single-stranded DNA flaps %K 40 nm diameter neutravidin bead %K single-molecule fluorescence microscopy %X 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. %I ACS Publications