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In the fluorometric experiments, trace amounts of the organic dye SYBR green (SG) were added to one- pot self-assembly reaction mixtures (18). The use of low concentrations of SG corresponding to one SG molecule per ~900 possible DNA base pairs in solution ensured negligible perturbation of the DNA hybridization processes. The fluorescence brightness of SG is enhanced upon intercalation into dsDNA domains (29); recording the SG fluorescence intensity can thus provide a measure for the evolution of the overall content of DNA base pairs in solution. A specific signal for the formation of object-related DNA base pairs upon incubation for time t at temperature T is obtained by comparison with reference reactions (18). For templated DNA objects-i.e., objects that form by staple DNA strand-assisted folding of a much longer scaffold DNA template molecule (4)-reference reactions were prepared that included the respective set of staple DNA oligonucleotides but lacked the scaffold DNA strand. Cryogenic shock-freezing of a reaction mixture in liquid nitrogen quenched folding and unfolding processes and preserved the state of the reaction at the time and temperature of freezing (18). After quick thawing, the solution content was analyzed using agarose gel electrophoresis and/or direct imaging with negative-stain TEM. Gel electrophoresis and TEM imaging were performed as previously described (30). Excess staple strands were removed by molecular weight cut-off filtration with 100-kD spin filters. Objects were designed using caDNAno (31); models were computed using CanDo (30, 32).
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